S-nitrosylated human serum albumin-mediated cytoprotective activity is enhanced by fatty acid binding

Abstract

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.

FIGURE 1.

Time profile of changes in serum levels of AST (A) and ALT (B) after hepatic ischemia/reperfusion in rats. Ischemia was induced by occluding both the portal vein and hepatic artery for 30 min. After that period of time reperfusion was established. Control (saline), 0.1 μmol of SNO-HSA per rat, or 0.1 μmol of SNO-HSA-OA (OA/HSA = 3 or 5) per rat was administered via the portal vein immediately after initiation of reperfusion. Blood was collected from the portal vein at various time points after reperfusion. ALT and AST activities were measured by using a sequential multiple AutoAnalyzer system. Data are expressed as means ± S.E. (n = 4 at each time point). ^*, p < 0.05 and ^**, p < 0.01, compared with control. #, p < 0.05, compared with the SNO-HSA-treated group. (OA/HSA = 3 or 5: HSA with 3 or 5 bound OA molecules per protein molecule.)

FIGURE 2.

Antiapoptotic effect of albumin without and with 5 mol OA/mol protein. HepG2 cells (2 × 10⁵ cells/well) were treated with different concentrations (0, 6.25, 12.5, 25, 50, 100 μm) of HSA or SNO-HSA, with and without bound OA, for 6 h at 37 °C in the dark. After incubation, the cells were washed three times with 10 mm phosphate-buffered saline, pH 7.4 to remove the remaining protein. Afterwards, the HepG2 cells were treated with anti-Fas antibody to induce apoptotic cell death. The number of apoptotic cells was determined by means of flow cytometry with annexin V-FITC and propidium iodide. Data are expressed as means ± S.E. (n = 6). ^*, p < 0.05 and ^**, p < 0.01, compared with the HSA-treated group. #, p < 0.05, ##, p < 0.01, compared with the SNO-HSA-treated group.

FIGURE 3.

Plasma concentrations of ¹¹¹In radioactivity (A) and tissue accumulation of radioactivity in liver (B), kidney (C), and spleen (D) after i.v. injection of SNO-HSA with different molar ratios of bound OA. ¹¹¹In-SNO-HSAs with and without OA were injected via the tail vein into male ddY mice at a dose of 0.1 mg/kg. At different times thereafter, mice were taken for collection of blood from the vena cava with the animal under ether anesthesia; plasma was obtained from the blood by centrifugation. After blood collection, the mice were euthanized, 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. Data are expressed as means ± S.E. (n = 3); the bars showing S.E. were smaller than the size of the symbols.

FIGURE 4.

Crystal structure of HSA showing locations of OA binding sites and the S-nitrosylated site (Cys-34) (A), and the effect of OA binding on the accessibility of Cys-34 (B). A, as seen, OA binding results in a more open protein structure. The subdivision of HSA into domains (I-III) is indicated. The structures were simulated on the basis of x-ray crystallographic data for HSA and HSA-OA (PDB ID codes 1bmo and 1gni, respectively) and modified with the use of Rasmol. B, SNO-HSA (300 μm), without and with different amounts of bound 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. Data are expressed as means ± S.E. (n = 4).

FIGURE 5.

S-Denitrosylation of SNO-HSA by GSH (A) and HepG2 cells (B). A, SNO-HSA (100 μm), without and with different amounts of bound OA, was incubated with GSH (100 μm) in 10 mm phosphate-buffered saline, pH 7.4 at 37 °C. After 0, 7.5, 15, and 30 min of incubation, samples were taken, and the concentrations of the remaining SNO-HSA (full curves) and the GS-NO formed (dotted curves) were determined separately by a HPLC flow reactor system. The T_½ values for the decline of SNO-HSAs are indicated by the arrows. B, SNO-HSA (100 μm), without and with different amounts of bound OA, was incubated with HepG2 cells (5 × 10⁵ cells/well) in 10 mm phosphate-buffered saline, pH 7.4 at 37 °C. After 0, 15, 30, or 60 min of incubation, the concentrations of the remaining SNO-HSA were determined by the HPLC flow reactor system. The T_½ values for the decline of SNO-HSAs are indicated. Data are expressed as means ± S.E. (n = 3). ^*, p < 0.05 and ^**, p < 0.01, compared with SNO-HSA without OA binding.

FIGURE 6.

S-Denitrosylation of SNO-HSA made from HSA isolated from hemodialytic patients before (A) and after (B) dialysis by HepG2 cells. HSA samples were obtained from hemodialytic patients before and after dialysis. The HSA samples were S-nitrosylated using IAN, as described above. HepG2 cells (5 × 10⁵ cells/well) and 100 μm SNO-HSA having different amounts of bound endogenous fatty acids were incubated in 10 mm phosphate-buffered saline, pH 7.4 at 37 °C. After 15, 30, and 60 min of incubation, the concentration of the remaining SNO-HSA was measured by the HPLC flow reactor system. The average T_½ for the S-denitrosylation is indicated, i.e. 48 min in A and 25 min in B. Data are expressed as means of four experiments. C, relationship between the individual T_½ values and the fatty acid contents of the HSA samples used. Samples are represented by open circles (before dialysis) or by closed circles (after dialysis); the colors correspond to the hemodialytic patients' number and color in panel A and panel B.

FIGURE 7.

Intracellular NO concentration of HepG2 cells exposed to SNO-HSA with different molar ratios of OA. The HepG2 cells (5 × 10⁵ cells/well) were first incubated with 5 μm DAF-FM DA for 1 h and then treated with 100 μm SNO-HSA with different amounts of bound OA in 10 mm phosphate-buffered saline, pH 7.4 in the dark at 37 °C. Some of the experiments with the highest OA concentration were also performed in the presence of 50 μm filipin III. Intracellular NO was monitored with DAF-FM fluorescence (ex/em of 385/535 nm). Δfluorescence represents DAF-FM fluorescence in cells incubated with different preparations of SNO-HSA, minus the fluorescence in cells that had been incubated with buffer only. Data are expressed as means ± S.E. (n = 4); the bars showing S.E. were smaller than the size of the symbols.

FIGURE 8.

Effect of OA and filipin III on binding of FITC-SNO-HSA to HepG2 cells. To prepare FITC-SNO-HSA, HSA was first S-nitrosylated as described above, followed by FITC labeling. FITC-SNO-HSA (50 μg/ml) with varying OA content (0, 3, 5 OA/HSA) was dissolved in 10 mm phosphate-buffered saline (pH 7.4) and added to HepG2 cells (5 × 10⁵ cells/well) for 10 min at 4 °C. In some experiments, the cells had been pretreated with 50 μm filipin III for 30 min. After incubation, the cells were washed twice with the phosphate-buffered saline to remove unbound FITC-SNO-HSA. After washing, the cells were analyzed using a fluorescence microscope. E, four columns quantify the FITC-fluorescence in panels A, B, C, and D, respectively, using the fluorescence in panel A as a reference value, i.e. 100%. The values are means ± S.E. (n = 3). ^*, p < 0.05 and ^**, p < 0.01, compared with the fluorescence of FITC-SNO-HSA without OA. #, p < 0.05, compared with the fluorescence of FITC-SNO-HSA with 3 mol of OA/HSA.

FIGURE 9.

Proposed model for the OA-mediated increase in S-transnitrosation of HepG2 cells by SNO-HSA. The S-transnitrosation can lead to cytoprotective effects, see Figs. 1 and 2. FA, free fatty acid.