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Antioxidant Function of the Mitochondrial Protein SP-22 in the Cardiovascular System

1999; Elsevier BV; Volume: 274; Issue: 4 Linguagem: Inglês

10.1074/jbc.274.4.2271

1083-351X

Masaru Araki, Hiroki Nanri, Kuniaki Ejima, Yoshinobu Murasato, Toshiyuki Fujiwara, Yasuhide Nakashima, Masaharu Ikeda,

Cholinesterase and Neurodegenerative Diseases

The mitochondrial protein SP-22 has recently been reported to be a member of the thioredoxin-dependent peroxide reductase family, suggesting that it may be one of the antioxidant systems in mitochondria, which are the major site of reactive oxygen intermediate generation. The aim of this study was to examine whether SP-22 is involved in mitochondrial antioxidant mechanisms and whether its expression is induced by oxidative stresses, particularly those in mitochondria. The expression of SP-22 protein was enhanced by about 1.5–4.6-fold when bovine aortic endothelial cells (BAEC) were exposed to various oxidative stresses, including mitochondrial respiratory inhibitors which increased the superoxide generation in BAEC mitochondria. The expression of SP-22 mRNA increased 2.0–3.5-fold with a peak at 3–6 h after exposure to Fe2+/dithiothreitol or a respiratory inhibitor, antimycin A. BAEC with an increased level of SP-22 protein caused by pretreatment with mild oxidative stress became tolerant to subsequent intense oxidative stress. On the other hand, BAEC that had been depleted of SP-22 with an antisense oligodeoxynucleotide against SP-22 mRNA became more labile to oxidative stress than control BAEC. The induction of SP-22 protein by oxidative stress in vivo was demonstrated in an experimental model of myocardial infarction in rat heart. These findings indicate that SP-22 functions as an antioxidant in mitochondria of the cardiovascular system. The mitochondrial protein SP-22 has recently been reported to be a member of the thioredoxin-dependent peroxide reductase family, suggesting that it may be one of the antioxidant systems in mitochondria, which are the major site of reactive oxygen intermediate generation. The aim of this study was to examine whether SP-22 is involved in mitochondrial antioxidant mechanisms and whether its expression is induced by oxidative stresses, particularly those in mitochondria. The expression of SP-22 protein was enhanced by about 1.5–4.6-fold when bovine aortic endothelial cells (BAEC) were exposed to various oxidative stresses, including mitochondrial respiratory inhibitors which increased the superoxide generation in BAEC mitochondria. The expression of SP-22 mRNA increased 2.0–3.5-fold with a peak at 3–6 h after exposure to Fe2+/dithiothreitol or a respiratory inhibitor, antimycin A. BAEC with an increased level of SP-22 protein caused by pretreatment with mild oxidative stress became tolerant to subsequent intense oxidative stress. On the other hand, BAEC that had been depleted of SP-22 with an antisense oligodeoxynucleotide against SP-22 mRNA became more labile to oxidative stress than control BAEC. The induction of SP-22 protein by oxidative stress in vivo was demonstrated in an experimental model of myocardial infarction in rat heart. These findings indicate that SP-22 functions as an antioxidant in mitochondria of the cardiovascular system. reactive oxygen intermediate 22-kDa substrate protein 23-kDa macrophage stress protein bovine aortic endothelial cells manganese superoxide dismutase 4-hydroxy-2-nonenal submitochondrial particles reverse transcription polymerase chain reaction digoxigenin dithiothreitol oligodeoxynucleotide copper and zinc superoxide dismutase superoxide dismutase tumor necrosis factor polyacrylamide gel electrophoresis fetal calf serum. Mitochondria play an important role in aerobic energy metabolism of living cells. The mitochondrial electron transport system consumes approximately 85% of the oxygen utilized by the cell, and about 5% of the oxygen is converted to reactive oxygen intermediates (ROIs)1 (1Forman H.J. Boveris A. Pryor W.A. Free Radicals in Biology. V. Academic Press, New York1982: 65-90Google Scholar, 2Shigenaga M.K. Hagen T.M. Ames B.N. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10771-10778Crossref PubMed Scopus (1831) Google Scholar). The generation of ROIs in mitochondria has been reported to impair various cellular functions by attacking reactive moieties of macromolecules such as protein, DNA, and lipid (3Stadtman E.R. Annu. Rev. Biochem. 1993; 62: 797-821Crossref PubMed Scopus (1265) Google Scholar, 4Cahill A. Wang X. Hoek J.B. Biochem. Biophys. Res. Commun. 1997; 235: 286-290Crossref PubMed Scopus (100) Google Scholar, 5Yakes F.M. Van H.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 514-519Crossref PubMed Scopus (1451) Google Scholar, 6Fantone J.C. Ward P.A. Am. J. Pathol. 1982; 107: 395-418PubMed Google Scholar). Mitochondrial permeability transition, which may be an initial event in the process of cell death induced by Ca2+ and inorganic phosphate, has recently been reported to be mediated by ROIs and prevented by antioxidants in vitro (7Kowaltowski A.J. Netto L.E. Vercesi A.E. J. Biol. Chem. 1998; 273: 12766-12769Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). The mitochondrial protein SP-22 was originally isolated from bovine adrenal cortex as a substrate protein for mitochondrial ATP-dependent protease (8Watabe S. Kohno H. Kouyama H. Hiroi T. Yago N. Nakazawa T. J. Biochem. (Tokyo). 1994; 115: 648-654Crossref PubMed Scopus (97) Google Scholar, 9Watabe S. Hasegawa H. Takimoto K. Yamamoto Y. Takahashi S.Y. Biochem. Biophys. Res. Commun. 1995; 213: 1010-1016Crossref PubMed Scopus (58) Google Scholar). An analysis of its amino acid sequence revealed that SP-22 is a member of the thioredoxin-dependent peroxide reductase family like the C22 component of alkyl hydroperoxidase in Salmonella typhimurium (10Tartaglia L.A. Storz G. Brodsky M.H. Lai A. Ames B.N. J. Biol. Chem. 1990; 265: 10535-10540Abstract Full Text PDF PubMed Google Scholar), thiol-specific antioxidant enzyme (11Chae H.Z. Chung S.J. Rhee S.G. J. Biol. Chem. 1994; 269: 27670-27678Abstract Full Text PDF PubMed Google Scholar), 23-kDa macrophage stress protein (MSP23) (12Ishii T. Yamada M. Sato H. Matsue M. Taketani S. Nakayama K. Sugita Y. Bannai S. J. Biol. Chem. 1993; 268: 18633-18636Abstract Full Text PDF PubMed Google Scholar), natural killer cell enhancing factor (NKEF) (13Shau H. Gupta R.K. Golub S.H. Cell Immunol. 1993; 147: 1-11Crossref PubMed Scopus (134) Google Scholar), and MER5 (14Yamamoto T. Matsui Y. Natori S. Obinata M. Gene (Amst). 1989; 80: 337-343Crossref PubMed Scopus (110) Google Scholar) in mammalian cells. MER5 is 92% similar to SP-22 protein and considered to be a mouse homolog of SP-22 (15Watabe S. Hiroi T. Yamamoto Y. Fujioka Y. Hasegawa H. Yago N. Takahashi S.Y. Eur. J. Biochem. 1997; 249: 52-60Crossref PubMed Scopus (132) Google Scholar). Members of this family have a highly conserved active site sequence among a wide range of species and are believed to act as antioxidant systems together with the NADPH-thioredoxin-thioredoxin reductase system (11Chae H.Z. Chung S.J. Rhee S.G. J. Biol. Chem. 1994; 269: 27670-27678Abstract Full Text PDF PubMed Google Scholar, 16Kang S.W. Baines I.C. Rhee S.G. J. Biol. Chem. 1998; 273: 6303-6311Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). Among the members of the thioredoxin-dependent peroxide reductase family, SP-22 is the only protein located in mitochondria. To test the hypothesis that SP-22 functions as an antioxidant system in mitochondria, which are the major site of cellular ROI generation, we investigated the oxidant-induced expression of SP-22 using cultured bovine aortic endothelial cells (BAEC) and an in vivo model of experimental myocardial infarction. Furthermore, we also examined the antioxidant function of SP-22 using BAEC with decreased or increased levels of SP-22 protein produced by treatment with antisense oligodeoxynucleotide or mild oxidant preconditioning. The present results indicate that SP-22 plays a crucial role in the antioxidant defense mechanism of mitochondria in the cardiovascular system. BAEC were harvested from bovine thoracic aorta obtained from a local slaughterhouse and cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) as described by Kaku et al. (17Kaku Y. Nanri H. Sakimura T. Ejima K. Kuroiwa A. Ikeda M. Biochim. Biophys. Acta. 1997; 1356: 43-52Crossref PubMed Scopus (41) Google Scholar). The identity of endothelial cells was verified by their characteristic morphology and the presence of factor VIII-associated antigen. Endothelial cells at up to passage 10 were used for these experiments. Mouse anti-manganese superoxide dismutase (MnSOD) monoclonal antibody was purchased from Chemicon International (Temecula, CA), anti-4-hydroxy-2-nonenal (HNE) monoclonal antibody was from NOF Co. (Tokyo, Japan), 1-methyl-4-phenylpyridinium was from Aldrich, paraquat from Nacalai Tesque (Kyoto, Japan), antimycin A and superoxide dismutase were from Sigma, and glucose oxidase and xanthine oxidase were from Boehringer Mannheim (Mannheim, Germany). Other chemicals were standard commercial products of analytical grade. Monolayers of confluent BAEC were incubated for the indicated periods with/without oxidative stress agents in Eagle's minimal essential medium (Nissui, Japan) containing 0.5% FCS at 37 °C. After washing the monolayers, cells were disrupted in 150 μl of lysis buffer (50 mm Tris/HCl buffer, pH 7.4, containing 0.2% (w/v) Triton X-100, 1 mmEDTA, 5 μg/ml chymostatin, 10 μg/ml each leupeptin, antipain, and pepstatin, and 20 μm(p-amidinophenyl)methanesulfonyl fluoride hydrochloride) and homogenized with a Teflon homogenizer. After centrifugation at 7000 × g for 10 min at 4 °C, lactate dehydrogenase activity in the supernatant was assayed as described by Bergmeyeret al. (17Kaku Y. Nanri H. Sakimura T. Ejima K. Kuroiwa A. Ikeda M. Biochim. Biophys. Acta. 1997; 1356: 43-52Crossref PubMed Scopus (41) Google Scholar). The peptide SPTASREYFEKVNQ, corresponding to residues 183–195 of the SP-22 protein (9Watabe S. Hasegawa H. Takimoto K. Yamamoto Y. Takahashi S.Y. Biochem. Biophys. Res. Commun. 1995; 213: 1010-1016Crossref PubMed Scopus (58) Google Scholar), was synthesized and conjugated with hemocyanin. Female Japanese white rabbits were subcutaneously immunized with the conjugated peptide (500 μg) emulsified with adjuvant (Titer Max, Sigma). The first booster injection (250 μg) was given 4 weeks later, and this was followed by three booster injections (250 μg each) at 2-week intervals. Sera were obtained 2 weeks after the last booster injection. For immunofluorescence microscopy, KB cells grown on coverslips in minimal essential medium/10% FCS were fixed with 4% paraformaldehyde in phosphate-buffered saline and permeabilized with 0.1% saponine. The permeabilized cells were reacted with anti SP-22 antibody and then stained with rhodamine-conjugated second antibody. Electron immunocytochemistry of bovine mitochondria was carried out as described previously (18Kang D. Nishida J. Iyama A. Nakabeppu Y. Furuichi M. Fujiwara T. Sekiguchi M. Takeshige K. J. Biol. Chem. 1995; 270: 14659-14665Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). The crude mitochondrial fraction of KB cells was pelleted, fixed with 8% paraformaldehyde in 0.1 m phosphate buffer, pH 7.4, and embedded in LR white resin at 50 °C. Thin sections of the mitochondria were incubated with the antibody against the SP-22, and then incubated with anti-rabbit IgG-gold. Immunolabeled sections were then stained with uranyl acetate and lead citrate and examined under a Hitachi HU 12 electron microscope at 100 kV. Control experiments done with preimmune serum gave no immunoreactive signals. BAEC submitochondrial particles (SMP) were prepared essentially as described by Kang et al. (18Kang D. Nishida J. Iyama A. Nakabeppu Y. Furuichi M. Fujiwara T. Sekiguchi M. Takeshige K. J. Biol. Chem. 1995; 270: 14659-14665Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Briefly, BAEC were washed in an isotonic sucrose buffer composed of 10 mmTris-HCl, pH 7.4, 1 mm EDTA, 0.25 m sucrose, 15 μg/ml leupeptin, 5 μg/ml (p-amidinophenyl)methanesulfonyl fluoride hydrochloride, and 50 ng/ml pepstatin, and suspended in the same buffer (1 × 108 cells/ml). The cells were homogenized in a Potter-Elvehjem homogenizer and centrifuged twice at 600 ×g for 10 min to obtain post-nuclear supernatant. The post-nuclear supernatant was centrifuged at 7000 × gfor 10 min. The pellet (crude mitochondrial fraction) was sonicated and centrifuged at 320,000 × g for 1 h. The resultant pellet was homogenized in an isotonic sucrose buffer and served as the SMP fraction. Superoxide production by SMP was determined by the oxidation of adrenaline to adrenochrome, and was corrected by subtracting the rate in the presence of 10 μg/ml superoxide dismutase as described by Takeshige et al. (19Takeshige K. Minakami S. Biochem. J. 1979; 180: 129-135Crossref PubMed Scopus (258) Google Scholar). SP-22 protein of BAEC was determined by immunoblotting. Proteins (20 μg) from each sample of BAEC were separated by 15% SDS-polyacrylamide gel electrophoresis (PAGE), electrotransferred onto Immobilon P (Millipore), and probed with anti-SP-22 serum. Immunoreactive proteins were detected using horseradish peroxidase-conjugated goat anti-rabbit antibody and POD Immunostain (Wako, Osaka, Japan). Semiquantitative RT-RCR was performed to quantitate SP-22 mRNAs in oxidant-treated BAEC, as described previously (17Kaku Y. Nanri H. Sakimura T. Ejima K. Kuroiwa A. Ikeda M. Biochim. Biophys. Acta. 1997; 1356: 43-52Crossref PubMed Scopus (41) Google Scholar). Briefly, total RNA was isolated from confluent BAEC using TRIzol reagent (Life Technologies, Inc.) based on the method reported by Chomczynski and Sacchi (20Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63167) Google Scholar). RT reactions were carried out with Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.). The reverse-transcribed cDNA products were amplified withTaq DNA polymerase in a PJ2000 DNA thermal cycler (Perkin-Elmer). The conditions for each cycle were 94 °C for 45 s, 55 °C for 1 min, and 72 °C for 2 min (31 cycles). The following gene-specific primers for SP-22 were used: 5′-tcacgatgtgaactgcgaagttg-3′(bases 366–388, sense) and 5′-ggatggggcttgattgtaggag-3′(bases 710–731, antisense), which amplified a 366-base pair product. Each primer set yielded a single PCR product of the predicted size. The identity of the PCR products was confirmed by direct cycle sequencing. RT-PCR was also performed for the housekeeping gene β-actin as a control for the amount of RNA used in the RT reaction. A negative control, in which reverse transcriptase was omitted, was also performed to exclude the possibility of the amplification of contaminating genomic DNA. Linear relationships were observed between the quantity of RNA subjected to the RT reaction and the amount of amplified PCR product under the PCR conditions used for SP-22 (31 cycles) and β-actin (24 cycles) (Fig.1). SP-22 PCR products (366 base pairs) were subcloned into the pGEM-T Easy Vector (Promega) by means of the TA cloning technique. After confirming the direction of the PCR insert by sequencing, the plasmids with inserts were linearized withBSP 120I (Fermentas Ltd., Vilnius, Lithuania) and transcribed in vitro into digoxigenin (DIG)-labeled cRNA with SP6 RNA polymerase using a DIG RNA labeling kit (Boehringer Mannheim). Total RNA (5 μg) was dissolved in 12 μl of sample buffer containing 10 mm sodium phosphate, pH 7.0, 50% (w/v) dimethyl sulfoxide, and 1 m glyoxal. After denaturation (1 h at 50 °C), the samples were electrophoresed in 1% agarose gel with a 10 mm sodium phosphate buffer, and then transferred to a nylon membrane (Boehringer Mannheim) and immobilized by incubation for 30 min at 121 °C. Hybridization with the DIG-labeled cRNA probe was carried out overnight at 68 °C in 500 mm sodium phosphate, 7% SDS, 1 mm EDTA, and 1 mg/ml yeast tRNA (21Church G.M. Gilbert W. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1991-1995Crossref PubMed Scopus (7266) Google Scholar). Blots were washed twice at room temperature with 200 mmsodium phosphate, 5% SDS, and 1 mm EDTA, and for 15 min at 65 °C with 0.2× SSC plus 0.2% SDS before color reaction using a DIG nucleic acid detection kit (Boehringer Mannheim) according to the instructions provided by the supplier. The amount of SP-22 mRNA was normalized by 18 S ribosomal RNA. BAEC were preincubated with 3.3 μm Fe2+, 330 μm dithiothreitol (DTT) for 24 h, which maximally increased the expression of SP-22 protein. These treated BAEC were then exposed to more intense oxidative stress consisting of 3.3 μm Fe2+, 1 mm DTT for 24 h, and cell viabilities were evaluated by measuring the lactate dehydrogenase activities of the cell lysate. A 20-mer antisense phosphorothioate oligodeoxynucleotide (ODN) was synthesized by Toagosei Inc. Japan. The first antisense ODN sequence (antisense 1; 5′-catcttcgttatgcagggct-3′) is directed against the translation initiation region of the SP-22 mRNA. The second antisense ODN (antisense 2; 5′-ccttcaccaagcggagggtc-3′) is directed against the internal region of SP-22 mRNA. We also prepared a sense ODN of antisense 1 and 2, and several random ODNs for a control experiment. All of these ODNs were designed to not possess sequences homologous to other genomic sequences or strong secondary structures (22Wagner R.W. Nature. 1994; 372: 333-335Crossref PubMed Scopus (804) Google Scholar). For efficient transfection of BAEC, we used the cationic lipid Tfx-50 reagent (Promega). ODNs that had been diluted in 30 mmHEPES buffer, pH 7.4 (final concentration of 3.3 μm), and 5 μl of Tfx-50 reagent (final concentration of 5 μm), which was prepared according to the instructions provided by the supplier, were mixed and incubated for 15 min at room temperature. The ODN/Tfx-50 reagent mixture was added to each plate with 1 ml of RPMI 1640 medium without FCS, and the cells were incubated for 1 h at 37 °C in a 5% CO2 atmosphere. At the end of the incubation period, the cells were gently overlaid with 2 ml of RPMI 1640 medium containing 10% FCS. This process was performed twice more at 24-h intervals. Adult male Wistar rats weighing about 250 g were used for these experiments. The experiments were performed in accordance with the guidelines specified for institutional animal care and use of the University of Occupational and Environmental Health, Japan. The animals were anesthetized and ventilated by a Harvard small animal respirator. The left descending coronary artery was ligated via a left-sided thoracotomy, which results in infarction of the free left ventricular wall (23Maclean D. Fishbein M.C. Braunwald E. Maroko P.R. J. Clin. Invest. 1978; 61: 541-551Crossref PubMed Scopus (143) Google Scholar). In a sham procedure, a superficial suture was placed in the epicardium of the left ventricle, near the left descending coronary artery. A total of 9 infarcted rats and 9 sham-operated rats (n = 3 per time point) were killed either 1 or 2 days after surgery. The heart was frozen in liquid nitrogen and stored at −80 °C. The hearts from the sham group and infarct group, at 1 or 2 days after the operation (n = 3), were cut and the sections were fixed with acetone for 5 min. Endogenous peroxidase was then blocked with 0.3% H2O2 in methanol. The thin sections were reacted with the anti-SP-22 polyclonal antibody and anti-HNE monoclonal antibody, and stained by a Vectastain® Elite ABC kit (Vector Laboratories, Burlingame, CA). The sections were counterstained with hematoxylin. Protein concentrations were determined by the Bio-Rad protein assay kit using bovine serum albumin as a standard. The intensity of the stained bands in immunoblotting, Northern blotting, and RT-PCR was quantitated by densitometric analysis using the public domain computer program NIH Image (Wayne Rasband, NIH, Research Service Branch, National Institute of Mental Health, Bethesda, MD), and the results are expressed as means ± S.E. A statistical analysis was performed using Student's t-test. Differences were considered statistically significant at p < 0.05. We prepared an antibody against SP-22 protein and used it for immunological analysis. In immunoblotting, this antibody detected a single band with a molecular mass corresponding to SP-22 (22 kDa) in total homogenate and the mitochondrial fraction of BAEC, but not in the cytosol fraction (Fig.2 A). Intracellular distribution of SP-22 protein was further examined by immunofluorescence microscopy. When cultured KB cells were stained with the anti-SP-22 antibody, the signals of immunoreactive SP-22 exhibited the mitochondrial staining pattern characterized by a reticular staining appearance (Fig. 2 B). Immunostaining signals were hardly visible when preimmune sera for SP-22 were used. The localization of the SP-22 protein in the mitochondria was further confirmed by electron microscopic immunocytochemistry. The mitochondrial fraction isolated from KB cells was stained with the anti-SP-22 antibody, followed by gold-labeled second antibody. Proteins reactive to the antibody were located in the mitochondria (Fig.2 C). These results indicate that SP-22 protein is located in mitochondria of culture cells, which is consistent with the results of the biochemical analysis by Watabe et al. (8Watabe S. Kohno H. Kouyama H. Hiroi T. Yago N. Nakazawa T. J. Biochem. (Tokyo). 1994; 115: 648-654Crossref PubMed Scopus (97) Google Scholar). Exposing BAEC to Fe2+/DTT, which produces the hydroxyl radical through the Fenton reaction (24Lesko S.A. Lorentzen R.J. Ts'o P.O. Biochemistry. 1980; 19: 3023-3028Crossref PubMed Scopus (251) Google Scholar, 25McCord J.M. Day E.J. FEBS Lett. 1978; 86: 139-142Crossref PubMed Scopus (666) Google Scholar), increased the expression of SP-22 protein in a time-dependent manner, with the maximal elevation occurring after 24 h of exposure. On the other hand, the expression of MnSOD was induced only slightly in this condition (Fig.3). SP-22 expression was completely suppressed with Fe chelators, such as deferoxamine and diethylene triamineacetic acid (Table I). TableII summarizes the effects of various oxidative stresses on the induction of SP-22 protein in BAEC. There was no apparent difference in the induction profile specific to ROI species. Peroxides such as hydrogen peroxide,tert-butylhydroperoxide, and cummene hydroperoxide enhanced SP-22 protein about 1.5–2.3-fold. Glucose oxidase and xanthine/xanthine oxidase, which produce hydrogen peroxide and superoxide (26Kwak H.S. Yim H.S. Chock P.B. Yim M.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4582-4586Crossref PubMed Scopus (40) Google Scholar, 27McCord J.M. Fridovich I. J. Biol. Chem. 1968; 243: 5753-5760Abstract Full Text PDF PubMed Google Scholar), enhanced SP-22 protein by about 40% increase. Sodium arsenite and cadmium chloride, which interact with sulfhydryl groups (28Levinson W. Oppermann H. Jackson J. Biochim. Biophys. Acta. 1980; 606: 170-180Crossref PubMed Scopus (260) Google Scholar), also enhanced SP-22 protein by about 50–100% increase. However, diethylmaleate and buthionine sulfoximine, which are glutathione depletors that have been shown to induce other thioredoxindependent peroxide reductase proteins (12Ishii T. Yamada M. Sato H. Matsue M. Taketani S. Nakayama K. Sugita Y. Bannai S. J. Biol. Chem. 1993; 268: 18633-18636Abstract Full Text PDF PubMed Google Scholar), exhibited a rather poor induction of SP-22 protein. Rotenone, paraquat, 1-methyl-4-phenylpyridinium, antimycin A, and KCN, which are respiratory chain inhibitors that increase ROI generation in mitochondria (29Freeman B.A. Crapo J.D. Lab. Invest. 1982; 47: 412-426PubMed Google Scholar), also enhanced SP-22 protein by 1.6–3.2-fold. ROI generation in SMP treated with antimycin A and rotenone showed 40–100% increase compared with untreated SMP (TableIII). Based on the rate of superoxide generation by antimycin A-treated SMP (4.1 nmol/min/mg protein), the antimycin A-treated confluent BAEC (107 cells) produced superoxide roughly at a rate of 0.13 nmol/min/mg protein (assuming that the protein content of SMP in 107 cells is about 0.065 mg), which is about 800-fold lower than the rate of superoxide generation produced by exogenous xanthine/xanthine oxidase (50 milliunits/ml), whereas the SP-22 protein induced by antimycin A treatment was about 3-fold higher than that induced by the xanthine/xanthine oxidase treatment, suggesting that superoxide production in mitochondria induced SP-22 expression more effectively.Table IInduction of SP-22 protein in BAEC exposed to the Fe2+/DTT systemStress agentsRelative SP-22 protein levelsFe2+ (3.3 μm)/DTT (330 μm)1.92 ± 0.19Fe2+ (3.3 μm)/DTT (500 μm)2.65 ± 0.53Fe2+ (3.3 μm)/DTT (1 mm)4.61 ± 0.83Fe2+ (3.3 μm)/DTT (330 μm) + DETAPAC (1 mm)0.92 ± 0.08Fe2+ (3.3 μm)/DTT (500 μm) + DETAPAC (1 mm)0.84 ± 0.13Fe2+ (3.3 μm)/DTT (1 mm) + DETAPAC (1 mm)0.93 ± 0.10DETAPAC (1 mm)0.85 ± 0.07Fe2+ (3.3 μm)/DTT (1 mm) + deferoxamine (1 mm)1.04 ± 0.26Deferoxamine (1 mm)0.64 ± 0.08BAEC were incubated for 24 h with Fe2+/DTT. Proteins of BAEC total homogenate were separated on 15% SDS-PAGE and immunoblotted with the anti-SP-22 antibody. Twenty μg of each protein were applied to each lane. Quantitative data were obtained by a densitometric analysis of the stained bands. The abundance of SP-22 protein is expressed as a ratio relative to that of the control BAEC (defined as 1.0). DTT, dithiothreitol; DETAPAC, diethylenetriamineacetic acid. Open table in a new tab Table IIInduction of SP-22 protein in BAEC exposed to various oxidative stressesStress agentsRelative SP-22 protein levelsH2O2 (500 μm)1.47 ± 0.19tert-Butylhydroperoxide (250 μm)1.82 ± 0.32Cummene hydroperoxide (500 μm)2.29 ± 0.63Glucose oxidase (10 milliunits/ml)1.44 ± 0.10Xanthine (50 μm)/xanthine oxidase (50 milliunits/ml)1.39 ± 0.05NaAsO2 (100 μm)1.89 ± 0.36CdCl2 (100 μm)1.69 ± 0.23Buthionine sulfoximine (100 μm)1.09 ± 0.09Diethylmaleate (50 μm)1.26 ± 0.16Rotenone (100 nm)1.75 ± 0.26Paraquat (330 μm)1.95 ± 0.22MPP+ (500 μm)2.49 ± 0.54Antimycin A (10 μm)a48-h incubation.3.23 ± 0.67KCN (1 mm)b12-h incubation.1.60 ± 0.162-Deoxyglucose (20 mm)1.03 ± 0.04BAEC were incubated with various oxidative stresses. Proteins of BAEC total homogenate were separated on 15% SDS-PAGE and immunoblotted with the anti-SP-22 antibody. Twenty μg of each protein were applied to each lane. Quantitative data were obtained by a densitometric analysis of the stained bands. The abundance of SP-22 protein is expressed as a ratio relative to that of the control BAEC (defined as 1.0). The data reflect the most significant increase in SP-22 protein after incubation. Incubation times were 24 h except with antimycin A and KCN. MPP+, 1-methyl-4-phenylpyridinium.a 48-h incubation.b 12-h incubation. Open table in a new tab Table IIIGeneration of superoxide by SMP treated with respiratory chain inhibitorsConditionsAdrenochrome formationnmol/min/mgControl system1.97 ± 0.02+ Rotenone (100 nm)2.76 ± 0.07+ Antimycin A (10 μm)4.01 ± 0.84SMP were prepared from BAEC as described under "Experimental Procedures." Superoxide production by SMP was determined by the oxidation of adrenaline to adrenochrome and corrected by subtracting the rate in the presence of 10 μg/ml superoxide dismutase, as described by Takeshige et al. (19Takeshige K. Minakami S. Biochem. J. 1979; 180: 129-135Crossref PubMed Scopus (258) Google Scholar). The control system contained 0.5 mg/ml SMP and 1 mm adrenaline, 0.25m sucrose, and 50 mm Hepes/NaOH, pH 7.5, in 1 ml. After preincubation for 5 min, the reaction was started under the conditions indicated adding of 0.2 mm NADPH, and adrenochrome formation was determined at 485–575 nm by a dual-wavelength spectrophotometer. The results represent the means ± S.E. of three separate experiments. Open table in a new tab BAEC were incubated for 24 h with Fe2+/DTT. Proteins of BAEC total homogenate were separated on 15% SDS-PAGE and immunoblotted with the anti-SP-22 antibody. Twenty μg of each protein were applied to each lane. Quantitative data were obtained by a densitometric analysis of the stained bands. The abundance of SP-22 protein is expressed as a ratio relative to that of the control BAEC (defined as 1.0). DTT, dithiothreitol; DETAPAC, diethylenetriamineacetic acid. BAEC were incubated with various oxidative stresses. Proteins of BAEC total homogenate were separated on 15% SDS-PAGE and immunoblotted with the anti-SP-22 antibody. Twenty μg of each protein were applied to each lane. Quantitative data were obtained by a densitometric analysis of the stained bands. The abundance of SP-22 protein is expressed as a ratio relative to that of the control BAEC (defined as 1.0). The data reflect the most significant increase in SP-22 protein after incubation. Incubation times were 24 h except with antimycin A and KCN. MPP+, 1-methyl-4-phenylpyridinium. SMP were prepared from BAEC as described under "Experimental Procedures." Superoxide production by SMP was determined by the oxidation of adrenaline to adrenochrome and corrected by subtracting the rate in the presence of 10 μg/ml superoxide dismutase, as described by Takeshige et al. (19Takeshige K. Minakami S. Biochem. J. 1979; 180: 129-135Crossref PubMed Scopus (258) Google Scholar). The control system contained 0.5 mg/ml SMP and 1 mm adrenaline, 0.25m sucrose, and 50 mm Hepes/NaOH, pH 7.5, in 1 ml. After preincubation for 5 min, the reaction was started under the conditions indicated adding of 0.2 mm NADPH, and adrenochrome formation was determined at 485–575 nm by a dual-wavelength spectrophotometer. The results represent the means ± S.E. of three separate experiments. Respiratory chain inhibitors also deplete ATP by blocking oxidative energy metabolism in mitochondria (30Loschen G. Azzi A. Richter C. Flohe L. FEBS Lett. 1974; 42: 68-72Crossref PubMed Scopus (504) Google Scholar, 31Hasegawa E. Takeshige K. Oishi