S-Nitrosylated Human Serum Albumin-mediated Cytoprotective Activity Is Enhanced by Fatty Acid Binding
2008; Elsevier BV; Volume: 283; Issue: 50 Linguagem: Inglês
10.1074/jbc.m807009200
ISSN1083-351X
AutoresYu Ishima, Takaaki Akaike, Ulrich Kragh‐Hansen, Shuichi Hiroyama, Tomohiro Sawa, Ayaka Suenaga, Toru Maruyama, Toshiya Kai, Masaki Otagiri,
Tópico(s)Mass Spectrometry Techniques and Applications
ResumoBinding of oleate to S-nitrosylated human serum albumin (SNO-HSA) enhances its cytoprotective effect on liver cells in a rat ischemia/reperfusion model. It enhances the antiapoptotic effect of SNO-HSA on HepG2 cells exposed to anti-Fas antibody. To identify some of the reasons for the increased cytoprotective effects, additional experiments were performed with glutathione and HepG2 cells. As indicated by 5,5′-dithiobis-2-nitrobenzoic acid binding, the addition of oleate increased the accessibility of the single thiol group of albumin. Binding of increasing amounts of oleate resulted in increasing and more rapid S-transnitrosation of glutathione. Likewise, binding of oleate, or of a mixture of endogenous fatty acids, improved S-denitrosation of SNO-HSA by HepG2 cells. Oleate also enhanced S-transnitrosation by HepG2 cells, as detected by intracellular fluorescence of diaminofluorescein-FM. All of the S-transnitrosation caused by oleate binding was blocked by filipin III. Oleate also increased, in a dose-dependent manner, the binding of SNO-HSA labeled with fluorescein isothiocyanate to the surface of the hepatocytes. A model in two parts was worked out for S-transnitrosation, which does not involve low molecular weight thiols. Fatty acid binding facilitates S-denitrosation of SNO-HSA, increases its binding to HepG2 cells and greatly increases S-transnitrosation by hepatocytes in a way that is sensitive to filipin III. A small nitric oxide transfer takes place in a slow system, which is unaffected by fatty acid binding to SNO-HSA and not influenced by filipin III. Thus, fatty acids could be a novel type of mediator for S-transnitrosation. Binding of oleate to S-nitrosylated human serum albumin (SNO-HSA) enhances its cytoprotective effect on liver cells in a rat ischemia/reperfusion model. It enhances the antiapoptotic effect of SNO-HSA on HepG2 cells exposed to anti-Fas antibody. To identify some of the reasons for the increased cytoprotective effects, additional experiments were performed with glutathione and HepG2 cells. As indicated by 5,5′-dithiobis-2-nitrobenzoic acid binding, the addition of oleate increased the accessibility of the single thiol group of albumin. Binding of increasing amounts of oleate resulted in increasing and more rapid S-transnitrosation of glutathione. Likewise, binding of oleate, or of a mixture of endogenous fatty acids, improved S-denitrosation of SNO-HSA by HepG2 cells. Oleate also enhanced S-transnitrosation by HepG2 cells, as detected by intracellular fluorescence of diaminofluorescein-FM. All of the S-transnitrosation caused by oleate binding was blocked by filipin III. Oleate also increased, in a dose-dependent manner, the binding of SNO-HSA labeled with fluorescein isothiocyanate to the surface of the hepatocytes. A model in two parts was worked out for S-transnitrosation, which does not involve low molecular weight thiols. Fatty acid binding facilitates S-denitrosation of SNO-HSA, increases its binding to HepG2 cells and greatly increases S-transnitrosation by hepatocytes in a way that is sensitive to filipin III. A small nitric oxide transfer takes place in a slow system, which is unaffected by fatty acid binding to SNO-HSA and not influenced by filipin III. Thus, fatty acids could be a novel type of mediator for S-transnitrosation. S-Nitrosothiols may serve as a reservoir of NO in biological systems, and they represent a class of NO donor with many potential biological and clinical uses. In this respect, Cys-34 of human serum albumin (HSA) 2The abbreviations used are: HSA, human serum albumin; SNO-HSA, S-nitrosylated HSA; GS-NO, S-nitrosylated GSH; OA, oleic acid/oleate; ALT, alanine aminotransferase; AST, asparatate aminotransferase; DAF-FM DA, diaminofluorescein-FM diacetate; FITC, fluorescein isothiocyanate; DTT, 1,4-dithiothreitol; DTNB, 5,5′-dithiobis-2-nitrobenzoic acid; DTPA, diethylenetriaminepentaacetic acid; IAN, isoamyl nitrite; DMEM, Dulbecco's modified Eagle's medium. is important, because it represents the largest fraction of free thiols in circulation (1.Peters Jr., T. All About Albumin: Biochemistry, Genetics, and Medical Applications. Academic Press, San Diego1996Google Scholar). In accordance with this proposal, S-nitrosylated HSA (SNO-HSA) has been reported to improve systolic and diastolic function, as well as myocardial perfusion and oxygen metabolism, in pigs during reperfusion after severe myocardial ischemia (2.Dworschak M. Franz M. Hallstrom S. Semsroth S. Gasser H. Haisjackl M. Podesser B.K. Malinski T. Pharmacology. 2004; 72: 106-112Crossref PubMed Scopus (20) Google Scholar, 3.Hallstrom S. Franz M. Gasser H. Vodrazka M. Semsroth S. Losert U.M. Haisjackl M. Podesser B.K. Malinski T. Cardiovasc. Res. 2008; 77: 506-514Crossref PubMed Scopus (34) Google Scholar) and to reduce ischemia/reperfusion injury in rabbit skeletal muscle (4.Hallstrom S. Gasser H. Neumayer C. Fugl A. Nanobashvili J. Jakubowski A. Huk I. Schlag G. Malinski T. Circulation. 2002; 105: 3032-3038Crossref PubMed Scopus (92) Google Scholar) and rat liver (5.Ishima Y. Sawa T. Kragh-Hansen U. Miyamoto Y. Matsushita S. Akaike T. Otagiri M. J. Pharmacol. Exp. Ther. 2007; 320: 969-977Crossref PubMed Scopus (57) Google Scholar). Among its other beneficial effects, SNO-HSA inhibits the activation of circulating platelets (6.Crane M.S. Ollosson R. Moore K.P. Rossi A.G. Megson I.L. J. Biol. Chem. 2002; 277: 46858-46863Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), suppresses apoptosis of human promonocytic cells (5.Ishima Y. Sawa T. Kragh-Hansen U. Miyamoto Y. Matsushita S. Akaike T. Otagiri M. J. Pharmacol. Exp. Ther. 2007; 320: 969-977Crossref PubMed Scopus (57) Google Scholar), and exhibits antibacterial activity in vitro (5.Ishima Y. Sawa T. Kragh-Hansen U. Miyamoto Y. Matsushita S. Akaike T. Otagiri M. J. Pharmacol. Exp. Ther. 2007; 320: 969-977Crossref PubMed Scopus (57) Google Scholar). HSA is a multifunctional protein synthesized and secreted by liver cells. It is a single, non-glycosylated polypeptide that organizes to form a heart-shaped protein with ∼67% α-helix but no β-sheet (1.Peters Jr., T. All About Albumin: Biochemistry, Genetics, and Medical Applications. Academic Press, San Diego1996Google Scholar). All but one (Cys-34) of the 35 cysteine residues are involved in the formation of stabilizing disulfide bonds. In circulation, approximately half of HSA contains Cys-34 as a free sulfhydryl, whereas the remainder is oxidized or ligand-bound. In addition to forming S-nitrosothiols, HSA can interact reversibly with a large number of endogenous and exogenous ligands (1.Peters Jr., T. All About Albumin: Biochemistry, Genetics, and Medical Applications. Academic Press, San Diego1996Google Scholar, 7.Kragh-Hansen U. Pharmacol. Rev. 1981; 33: 17-53PubMed Google Scholar, 8.Kragh-Hansen U. Chuang V.T.G. Otagiri M. Biol. Pharm. Bull. 2002; 25: 695-704Crossref PubMed Scopus (772) Google Scholar). Thus, one of the important in vivo functions of albumin is to transport fatty acids, and usually the protein carries different fatty acid anions, up to a total amount of 1–2 molar equivalents. However, this value can rise to about 4 after maximal exercise or other adrenergic stimulation (1.Peters Jr., T. All About Albumin: Biochemistry, Genetics, and Medical Applications. Academic Press, San Diego1996Google Scholar). In the present work, the effect of oleate (OA) on S-transnitrosation from SNO-HSA was studied. OA was used as a representative for the endogenous fatty acids, because quantitatively it is the most important fatty acid in human depot fat, and because it is a major contributor to the albumin-bound fatty acids. First, it was observed that co-binding of OA improved SNO-HSA-mediated cytoprotection against ischemia/reperfusion liver injury in rats and improved its antiapoptotic effect on human hepatocellular carcinoma (HepG2) cells exposed to anti-Fas antibody. In an attempt to explain these effects, S-transnitrosation was then investigated in simpler systems, namely from SNO-HSA to glutathione (GSH) and to HepG2 cells. The effect of a mixture of endogenous fatty acids on the latter S-transnitrosation was also studied. The influence of OA binding on NO transfer to the HepG2 cells and on the interaction between SNO-HSA and the hepatocytes was examined. Finally, a model for the OA-induced improvement of NO transfer to HepG2 cells is proposed. Materials—Non-defatted HSA (96% pure) was donated by the Chemo-Sera-Therapeutic Research Institute (Kumamoto, Japan), and it was defatted by treatment with charcoal as described by Chen (9.Chen R.F. J. Biol. Chem. 1967; 242: 173-181Abstract Full Text PDF PubMed Google Scholar). Sephadex G-25 (ϕ 1.6 × 2.5 cm) and Blue Sepharose CL-6B (ϕ 2.5 × 20 cm) were from GE Healthcare (Tokyo, Japan). OA, caprylate, stearate, GSH, 1,4-dithiothreitol (DTT), and filipin III were purchased from Sigma-Aldrich. Isoamyl nitrite (IAN) was purchased from Wako Chemicals (Osaka, Japan). Sulfanilamide, naphthylethylenediamine-hydrochloride, HgCl2, and NaNO2 were obtained from Nakalai Tesque (Kyoto, Japan). Diethylenetriaminepentaacetic acid (DTPA), 5,5′-dithiobis-2-nitrobenzoic acid (DTNB), and fluorescein isothiocyanate (FITC) were bought from Dojindo Laboratories (Kumamoto, Japan). Dulbecco's modified Eagle's medium (DMEM) was from Invitrogen (Rockville, MD), and diaminofluorescein-FM diacetate (DAF-FM DA) was from Daiichi Pure Chemicals (Tokyo, Japan). 111InCl3 (74 MBq/ml in 0.02 n HCl) was a gift from Nihon Medi-Physics Co., Ltd. (Hyogo, Japan). The other chemicals were of the best grades commercially available, and all solutions were made in deionized and distilled water. S-Nitrosylation of HSA—S-Nitrosylated protein was prepared with protection against light and according to previously reported methods (10.Akaike T. Inoue K. Okamoto T. Nishino H. Otagiri M. Fujii S. Maeda H. J. Biochem. (Tokyo). 1997; 122: 459-466Crossref PubMed Scopus (73) Google Scholar, 11.Ikebe N. Akaike T. Miyamoto Y. Hayashida K. Yoshitake J. Ogawa M. Maeda H. J. Pharmacol. Exp. Ther. 2000; 295: 904-911PubMed Google Scholar). First, HSA (300 μm) was incubated with DTT (molar ratio, protein:DTT = 1:10) for 5 min at 37 °C. After incubation, DTT was immediately removed by Sephadex G-25 gel filtration and eluted with 0.1 m potassium phosphate buffer (pH 7.4) containing 0.5 mm DTPA. Samples of 0.1 mm DTT-treated protein (0.8 mol sulfhydryl groups/mol protein) were then incubated with IAN (molar ratio, protein:IAN = 1:10) in 0.1 m potassium phosphate buffer (pH 7.4) containing 0.5 mm DTPA for 60 min at 37 °C. The amount of the S-nitroso moiety of SNO-HSA was quantified by HPLC coupled with a flow-reactor system, as previously reported (10.Akaike T. Inoue K. Okamoto T. Nishino H. Otagiri M. Fujii S. Maeda H. J. Biochem. (Tokyo). 1997; 122: 459-466Crossref PubMed Scopus (73) Google Scholar). The HPLC column was a gel filtration column for S-nitrosylated proteins (ϕ 8 × 300 mm), Diol-120, YMC, Kyoto, Japan. Briefly, the eluate from the HPLC column was mixed with a HgCl2 solution to decompose S-nitrosylated compounds to yield NO2- (via NO+). The NO2- generated was then detected after reaction with Griess reagent in the flow-reactor system. Controls performed in the absence of HgCl2 gave no protein-derived absorbency at 540 nm after reaction with the Griess reagent. Therefore, non-covalent association of the nitrite anion with albumin can be excluded. The S-nitrosylated product (0.35 ± 0.04 mol SNO-groups/mol protein; mean ± S.E., n = 53) was purified by Sephadex G-25 gel filtration, eluted with 0.1 m potassium phosphate buffer (pH 7.4) containing 0.5 mm DTPA, and concentrated by ultrafiltration (cutoff size of 7500 Da). These samples were stored at -80 °C until use. The protein content of all protein preparations used in this study was determined using the Bradford assay. Binding of Fatty Acid to HSA—A stock solution of 20 mm OA was made in methanol-H2O (1:1, v/v). Aliquots of the OA stock solution were added to HSA or SNO-HSA and dissolved in 0.1 m potassium phosphate buffer (pH 7.4) to give OA:HSA molar ratios of 1, 3, or 5; the maximal methanol concentration in the final solutions was 1.25%. Before use, the solutions were held at 37 °C for 30 min. The amount of bound OA was checked using the following approach. The solutions containing albumin and OA were applied to a Sephadex G-25 gel filtration column, the protein-containing fractions were pooled, and both the protein and OA concentrations in the pooled material were determined. The OA concentrations were determined by a colorimetric method using a commercial kit (WAKO NEFA kit) according to the manufacturer's instructions. Three standards (0.5, 1.0, and 1.97 mEq/liter) supplied by the vendor were used to establish a standard curve for determination of the concentration of OA. Cytoprotective Effect of SNO-HSAs in Vivo—A rat ischemia/reperfusion liver injury model was used to investigate the cytoprotective effect of SNO-HSA, as previously reported (5.Ishima Y. Sawa T. Kragh-Hansen U. Miyamoto Y. Matsushita S. Akaike T. Otagiri M. J. Pharmacol. Exp. Ther. 2007; 320: 969-977Crossref PubMed Scopus (57) Google Scholar, 11.Ikebe N. Akaike T. Miyamoto Y. Hayashida K. Yoshitake J. Ogawa M. Maeda H. J. Pharmacol. Exp. Ther. 2000; 295: 904-911PubMed Google Scholar). Male Wistar rats weighing between 200 and 230 g (Kyudo, Inc., Kumamoto, Japan) were used. The animals were fasted for 9 h before surgery, but were allowed access to water. The rats were anesthetized with ether during the operation. After the abdomen was shaved and disinfected with 70% ethanol, a complete midline incision was made. The portal vein and hepatic artery were exposed and cross-clamped for 30 min with a noncrushing microvascular clip. Saline, as the vehicle control, or HSA or SNO-HSA (0.1 μmol/rat) with or without bound OA was given via the portal vein immediately after reperfusion was initiated. Because the blood volume of a 200-g rat was estimated to be about 10 ml, we expected blood levels of SNO-HSA to reach ∼3 μm after administration of 0.1 μmol/rat of SNO-HSA. This concentration of SNO-HSA is a physiologically relevant one, because levels of S-nitrosothiols in normal plasma are at the most 7 μm (12.Wang X. Tanus-Santos J.E. Reiter C.D. Dejam A. Shiva S. Smith R.D. Hogg N. Gladwin M.T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11477-11482Crossref PubMed Scopus (133) Google Scholar). The abdomen was then closed in two layers with 2–0 silk. The rats were kept under warming lamps until they awakened and became active. Because blood loss caused by frequent blood sampling could affect liver function, the animals were euthanized by taking whole circulating blood via the abdominal aorta under anesthesia at various time points after reperfusion was initiated. Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured by using a sequential multiple AutoAnalyzer system from Wako Chemicals, with activities expressed in international units per liter. All the animal experiments were carried out according to the guidelines of the Laboratory Protocol of Animal Handling, Graduate School of Medical Sciences, Kumamoto University. Antiapoptotic Effect of SNO-HSA in Vitro—HepG2 cells (2 × 105 cells/well) were cultured in 96-well plates (16-mm diameter; Falcon, Lincoln Park, NJ) with DMEM supplemented with 10% fetal bovine serum (Invitrogen). The cells were maintained in a humidified incubator (95% air, 5% CO2) for 12 h at pH 7.4 and 37 °C. Afterwards, they were treated with different concentrations of HSA or SNO-HSA, with and without bound OA, for 6 h in the dark. The cells were then washed three times with 10 mm phosphate-buffered saline (pH 7.4) to remove the remaining SNO-HSA. After washing, the cells were reacted with 400 ng/ml anti-Fas antibody (Medical and Biological Laboratories, Nagoya, Japan). After 15 h of incubation, the cultures were treated with 0.05% trypsin, and the cells were transferred to Eppendorf tubes (1.5 ml). The number of apoptotic cells was determined by an annexin V-FITC binding assay kit from BD Biosciences (Tokyo, Japan). The fluorescence of annexin V-FITC and propidium iodide were measured by a FACSCalibur flow cytometer. Pharmacokinetic Experiments—SNO-HSA, with and without OA, was labeled with 111In, using DTPA anhydride as a bifunctional chelating agent (13.Hnatowich D.J. Layne W.W. Childs R.L. Int. J. Appl. Radiat. Isot. 1982; 33: 327-332Crossref PubMed Scopus (338) Google Scholar, 14.Yamasaki Y. Sumimoto K. Nishikawa M. Yamashita F. Yamaoka K. Hashida M. Takakura Y. J. Pharmacol. Exp. Ther. 2002; 301: 467-477Crossref PubMed Scopus (57) Google Scholar). Labeled proteins were injected via the tail vein into male ddY mice (weighing 25–27 g) at a dose of 0.1 mg/kg. At appropriate times after injection, blood was collected from the vena cava with the mouse under ether anesthesia. Heparin sulfate was used as an anticoagulant, and plasma was obtained from the blood by centrifugation. Liver, kidney, and spleen samples were obtained, rinsed with saline, and weighed. The radioactivity in each sample was counted using a well-type NaI scintillation counter ARC-2000 (Aloka, Tokyo, Japan). 111In radioactivity concentrations in plasma were normalized as a percentage of the dose per milliliter and analyzed using the nonlinear least-squares program MULTI (15.Yamaoka K. Tanigawara Y. Nakagawa T. Uno T. J. Pharmacobiodyn. 1981; 4: 879-885Crossref PubMed Scopus (2684) Google Scholar). Organ distribution profiles were evaluated by relating the radioactivity per gram of tissue to the total amount of injected radioactivity. Accessibility of Cys-34—We determined the accessibility of Cys-34 in reduced HSA with Ellman's reagent, DTNB (16.Gryzunov Y.A. Arroyo A. Vigne J.L. Zhao Q. Tyurin V.A. Hubel C.A. Gandley R.E. Vladimirov Y.A. Taylor R.N. Kagan V.E. Arch. Biochem. Biophys. 2003; 413: 53-66Crossref PubMed Scopus (90) Google Scholar, 17.Ishima Y. Akaike T. Kragh-Hansen U. Hiroyama S. Sawa T. Maruyama T. Kai T. Otagiri M. Biochem. Biophys. Res. Commun. 2007; 364: 790-795Crossref PubMed Scopus (27) Google Scholar). HSA (300 μm), with and without OA, and DTNB (5 mm) were mixed in 0.1 m potassium phosphate buffer (pH 7.0) at 20 °C, and the absorbance at 450 nm was registered as a function of time. S-Denitrosylation of SNO-HSA by GSH and by HepG2 Cells—Solutions with both 100 μm SNO-HSA and 100 μm GSH were made in 10 mm phosphate-buffered saline, pH 7.4. Then 0-, 7.5-, 15-, and 30-min samples were taken, mixed with 1/10 volume of 5 mm DTPA (pH 7.4) and placed at -80 °C. The concentrations of the remaining SNO-HSA and the S-nitrosylated GSH (GS-NO) formed were then determined separately by the HPLC flow reactor system. HepG2 cells (5 × 105 cells/well) were cultured and incubated with DMEM and fetal bovine serum, as described above. After incubation, the culture medium was removed, and the hepatocytes were washed three times with 10 mm phosphate-buffered saline, pH 7.4. Cells were further incubated at 37 °C in the CO2 incubator with 200 μl of 10 mm phosphate-buffered saline, pH 7.4 and 100 μm SNO-HSA with different molar ratios of bound OA. Samples were taken after incubation for 0, 15, 30, or 60 min, mixed with 1/10 volume of 5 mm DTPA, pH 7.4 and centrifuged at 10,000 × g for 10 min at 4 °C. These supernatants were stored at -80 °C until applied to the HPLC flow reactor system. HSA Obtained from Hemodialysis Patients—It is known that fatty acids bound to HSA rise by dialysis. The content of fatty acids bound to HSA isolated from hemodialysis patients before and after dialysis was analyzed, and the influence on S-denitrosation of fatty acid binding was examined. HSA samples were obtained from hemodialysis patients before (HSA-hd (-)) and after dialysis (HSA-hd (+)) according to a previously reported protocol (18.Watanabe H. Yamasaki K. Kragh-Hansen U. Tanase S. Harada K. Suenaga A. Otagiri M. Pharm. Res. 2001; 18: 1775-1781Crossref PubMed Scopus (53) Google Scholar). In brief, albumin concentrations in blood plasma were measured using a diagnostic kit (Biotech Reagent) based on the bromcresol green method. Non-esterified fatty acids were measured using a diagnostic kit from Wako Chemicals (Osaka, Japan). To isolate HSA from patient sera, polyethylene glycol fractionation of blood plasma was followed by chromatography on a Blue Sepharose CL-6B column. After isolation, the samples were dialyzed against deionized water for 48 h at 4 °C, followed by lyophilization. The purity of the HSA-hd samples was at least 95%, and the percentage of dimers did not exceed 7%, as evidenced by SDS-PAGE and native PAGE, respectively. The protocol used in this study was approved by the Institutional Review Board, and informed consent was obtained from all subjects. Preparation of S-Nitrosylated HSA-hd—HSA-hd (-) or HSA-hd (+) was S-nitrosylated using IAN, as described above. The content of SNO groups in SNO-HSA-hd was determined to be (0.35 ± 0.04 for HSA-hd (-) or 0.39 ± 0.06 for HSA-hd (+) mol SNO-groups/mol protein; mean ± S.E., n = 7). S-Denitrosylation of Hemodialysis Patient SNO-HSA by HepG2 Cells: Effects of HSA-bound Fatty Acids—One-hundred micromolar of SNO-HSA-hd (-) and SNO-HSA-hd (+) dissolved in phosphate-buffered saline was incubated with HepG2 cells (5 × 105 cells/well). After 15-, 30-, and 60-min incubation, culture supernatant was collected, and the remaining SNO content was measured and compared with control SNO-HSA samples before incubation. NO Uptake by HepG2 Cells—HepG2 cells (5 × 105 cells/well) were cultured and incubated with DMEM and fetal bovine serum, as described above. After incubation, the culture medium was removed, and the hepatocytes were washed three times with 10 mm phosphate-buffered saline, pH 7.4. After adding phosphate-buffered saline containing 5 μm DAF-FM DA, the cells were incubated for 1 h in the dark at 37 °C. The cells were again washed three times with phosphate-buffered saline, and further incubated at 37 °C with 100 μm HSA or SNO-HSA with different molar ratios of bound OA. Upon cellular uptake, ester groups in DAF-FM DA are cleaved by esterases, resulting in the formation of DAF-FM that can react with NO to give fluorescence. The fluorescence was determined with excitation at 385 nm and monitored at 535 nm using a monochromator (TECAN SPECTRA FLUOR). In some experiments, 50 μm filipin III (caveolae formation inhibitor) dissolved in phosphate-buffered saline was added after 30 min of DAF-FM DA exposure (19.Pohl J. Ring A. Stremmel W. J. Lipid Res. 2002; 43: 1390-1399Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Binding of FITC-HSA to HepG2 Cells—The binding of HSA to HepG2 cells, with or without S-nitrosylation and its modulation by fatty acid binding, was analyzed by means of fluorescent microscopy with HSA labeled with FITC. FITC-HSA was prepared according to a previous report (20.Maeda H. Ishida N. Kawauchi H. Tuzimura K. J. Biochem. (Tokyo). 1969; 65: 777-783Crossref PubMed Scopus (172) Google Scholar). In short, HSA (60 μm) was incubated with FITC (2 mm) for 3 h at 25 °C in 0.1 m potassium phosphate buffer (pH 7.4). After incubation, FITC-HSA was isolated from unreacted FITC using a Sephadex G-25 gel with the phosphate buffer, and stored at -80 °C until use. To prepare FITC-SNO-HSA, HSA was first S-nitrosylated as described above, followed by FITC labeling. After purification of FITC-SNO-HSA, the SNO content was determined to be (0.33 ± 0.03 mol SNO-groups/mol protein; mean ± S.E., n = 3), suggesting that FITC labeling did not affect the SNO content to a significant extent. For the binding assay, HepG2 cells (5 × 105 cells/well) were preincubated with serum-free DMEM for 2 h at 37 °C. In some experiments, cells were further treated with 50 μm filipin III for 30 min, which inhibits caveolae formation. Cells were then maintained at 4 °C to block the uptake of macromolecules through energy-dependent mechanisms, including endocytosis. FITC-SNO-HSA with varying OA content (0, 3, 5 OA/HSA) was dissolved in 10 mm phosphate-buffered saline, pH 7.4 and added to the culture wells to give a final concentration of 50 μg/ml. After 10 min, the cells were washed twice with phosphate-buffered saline to remove unbound FITC-SNO-HSA and fixed with 4% paraformaldehyde in phosphate-buffered saline at 25 °C for 30 min. After washing twice in MilliQ water, these cells were analyzed using a fluorescence microscope (Biozero-8000, Keyence, Osaka, Japan) with the combination of excitation at 385 nm and emission at 535 nm. Fluorescent intensity was quantified using an NIH Image. Similar experiments were conducted using FITC-HSA without S-nitrosylation. Statistical Analysis—The statistical significance of the collected data were evaluated using analysis of variance, followed by the Newman-Keuls method for more than 2 means. Data are expressed as means ± S.E. Differences between groups were evaluated using a Student's t test. p < 0.05 was regarded as statistically significant. Effect of OA Binding on SNO-HSA-mediated Cytoprotection against Ischemia/Reperfusion Liver Injury in Rats—An ischemia/reperfusion liver injury model (5.Ishima Y. Sawa T. Kragh-Hansen U. Miyamoto Y. Matsushita S. Akaike T. Otagiri M. J. Pharmacol. Exp. Ther. 2007; 320: 969-977Crossref PubMed Scopus (57) Google Scholar) was used to examine the in vivo effect of OA binding on SNO-HSA-mediated cytoprotection. Because previous studies with SNO-HSA showed that a quantity of 0.1 μmol/rat had the greatest protective effect (5.Ishima Y. Sawa T. Kragh-Hansen U. Miyamoto Y. Matsushita S. Akaike T. Otagiri M. J. Pharmacol. Exp. Ther. 2007; 320: 969-977Crossref PubMed Scopus (57) Google Scholar), the same quantity was used in this study. To evaluate liver injury, the extracellular release of the liver enzymes AST and ALT was measured via plasma enzyme values. Injecting HSA, OA, or HSA-OA into the portal vein immediately after reperfusion was initiated did not affect the plasma concentrations of AST and ALT (data not shown). However, administration of SNO-HSA diminished, to a significant extent, the enzyme concentrations measured after 60 and 120 min (Fig. 1). The protection of the liver cells by SNO-HSA was more pronounced if the protein also carried OA; the additive effect was most evident after 120 min of reperfusion. The effect of OA on SNO-HSA-mediated cytoprotection appears to depend on OA content, e.g. AST release was reduced significantly more by treatment with SNO-HSA-OA5 than with SNO-HSA-OA3 120 min after reperfusion (Fig. 1A). Even at 12 h after reperfusion, the levels of AST and ALT remained significantly lower in SNO-HSA-OA-treated animals than in control animals, although the differences were not so pronounced as those observed at 120 min after reperfusion (data not shown). We also examined the effect of other fatty acid on cytoprotective effect for SNO-HSA. In the ischemia/reperfusion injury model, we found that caprylate (C8:0), a short-chain and saturated fatty acid, potentiated the cytoprotective effect of SNO-HSA. The potentiation effect of caprylate was slightly weaker than that of OA. AST levels at 120 min after reperfusion were 3256 ± 55, 2586 ± 105, 2202 ± 253 for treatment with SNO-HSA, SNO-HSA-caprylate (HSA: caprylate = 1:5), SNO-HSA-OA5, respectively (The values are means ± S.E., n = 4). Effect of OA Binding on SNO-HSA-mediated Cytoprotection of HepG2 Cells Exposed to Anti-Fas Antibody—NO and related species reportedly induce both antiapoptotic and proapoptotic responses in cells, the type of response depending on the concentration of the NO donors and on the type of cell and apoptosis-inducing reagent (5.Ishima Y. Sawa T. Kragh-Hansen U. Miyamoto Y. Matsushita S. Akaike T. Otagiri M. J. Pharmacol. Exp. Ther. 2007; 320: 969-977Crossref PubMed Scopus (57) Google Scholar). In the present study, the influence of OA binding on the antiapoptotic effect of SNO-HSA on HepG2 cells treated with anti-Fas antibody was examined. As seen in Fig. 2, the presence of HSA, with or without bound OA, had no effect on the induced apoptosis. OA alone possessed no antiapoptotic activity in our HepG2 cell study (data not shown). By contrast, the addition of SNO-HSA resulted in concentration-dependent protection of the cells. This protection was greatly increased by binding 5 mol of OA per mol of SNO-HSA. Thus, fatty acid binding can also improve the cytoprotective effect of SNO-HSA in an in vitro system. Pharmacokinetic Experiments—The pharmacokinetic characteristics of SNO-HSA with and without bound OA were determined in mice. The results in Fig. 3A indicate that OA binding did not affect the elimination of SNO-HSA from the circulation. The plasma half-lives were 269.3 ± 1.7 min, 270.6 ± 1.2 min, and 271.6 ± 5.5 min for SNO-HSA, SNO-HSA-OA3, and SNO-HSA-OA5, respectively. Likewise, the uptake of SNO-HSA by liver, kidney, and spleen was unaffected by OA binding (Fig. 3, B–D). These data suggest that binding of as much as 5 mol of OA per mol of protein does not modify the pharmacokinetic properties of SNO-HSA. Accessibility of Cys-34 in HSA-OA—Crystallographic studies have revealed the existence of seven OA binding sites in HSA, and showed that none of these sites involve Cys-34 (21.Petitpas I. Grune T. Bhattacharya A.A. Curry S. J. Mol. Biol. 2001; 314: 955-960Crossref PubMed Scopus (436) Google Scholar) (Fig. 4A). Cys-34 is located in a crevice on the surface of the protein in subdomain IA (1.Peters Jr., T. All About Albumin: Biochemistry, Genetics, and Medical Applications. Academic Press, San Diego1996Google Scholar). Therefore, the improvement effect of even the highest OA concentration on the cytoprotection of SNO-HSA must be caused by indirect means. To test this hypothesis, the effect of OA on the readiness o
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