Eagle’s MEM, nonessential amino acids, and antibiotics were from Life Technologies Inc. (Grand Island, New York, USA). FBS was from HyClone Laboratories (Logan, Utah, USA). Glucosamine, wortmannin, and monoclonal anti-phosphoserine antibodies were purchased from Sigma Chemical Co. (St. Louis, Missouri, USA). Polyclonal anti–glutamine:fructose-6-phosphate amidotransferase (GFAT) antiserum was a kind gift of Erwin Schleicher (Tubingen, Germany). Monoclonal anti–O-linked GlcNAc antibodies (RL2) were from Affinity BioReagents Inc. (Golden, Colorado, USA). Anti–phospho-eNOS(Ser1177) was from Cell Signaling Technology Inc. (Beverly, Massachusetts, USA). Anti-eNOS IgG was from Santa Cruz Biotechnology Inc. (Santa Cruz, California, USA). Polyclonal antimitochondrial antibody was from Upstate Biotechnology Inc. (Lake Placid, New York, USA). Protein A–Sepharose, [3H]L-arginine monohydrochloride, poly(dI-dC), and poly dC were obtained from Amersham Pharmacia Biotech (Piscataway, New Jersey, USA).
Bovine aortic endothelial cells (BAECs; passages 4–10) were cultured to confluence in Eagle’s MEM containing 10% FBS, essential and nonessential amino acids, and antibiotics. Cells were incubated with either 5 mM glucose, 5 mM glucose plus 5 mM glucosamine, 10 mM glucosamine, or 500 nM wortmannin, as indicated; or with 30 mM glucose, 30 mM glucose plus GFAT oligonucleotides (antisense, inverse, or scrambled), uncoupling protein-1 (UCP-1), or manganese superoxide dismutase (MnSOD) recombinant adenovirus, as indicated. Experiments were also performed in which cells overexpressing UCP-1 or MnSOD were cultured in 30 mM glucose plus 10 mM glucosamine. Cells were transfected with plasmids encoding either myc-tagged wild-type human eNOS or myc-tagged human eNOS mutated at Ser1177 (mt S1177A) and incubated in either 5 mM glucose or 30 mM glucose, as indicated.
Oligonucleotide synthesis and treatment of cells.
Phosphorothioate oligonucleotides were synthesized by Operon Technologies Inc. (Alameda, California, USA). The S-antisense GFAT had the following sequence: 5′-cCACCTGCAAGACCATcG-3′ (15
). Inverse and scrambled oligonucleotides were used as controls. Oligonucleotide (36.3 μl) was mixed with 16.3 μl polyethyleneimine per ml of media (16
) to give a final concentration of oligonucleotide of 12.5 μM. Three hundred microliters of this solution was added to each well containing 200,000 cells. The cells were incubated in a CO2
incubator at 37°C for 2 hours. The oligonucleotide solution was then removed, the cells were washed with PBS, and fresh media was added. The cells were analyzed for eNOS activity 48 hours later.
Rat sense and antisense UCP-1 cDNA was provided by D. Riquier (Centre Nationale de la Recherche Scientifique, Unite Propre 1511, Meudon, France). Human MnSOD cDNA was provided by L. Oberley, University of Iowa College of Medicine. These cDNAs were cloned into the shuttle vector Ad5CMVK-NpA. Adenoviral vectors were prepared by the University of Iowa Gene Transfer Vector Core. Two microliters of either adenoUCP-1 or adenoMnSOD was added to 200,000 cells in a 24-well plate (moi = 500). The cells were incubated in a CO2 incubator at 37°C for 90 minutes. The media containing the virus was then removed and fresh media was added.
eNOS activity in cells was determined as previously described (17
). In brief, cells were first incubated in L
-arginine–deficient, serum-free MEM media for 6 hours. This media was then replaced with PBS buffer containing 120 mM NaCl, 4.2 mM KCl, 2.5 mM CaCl2
, 1.3 mM MgSO4
, 1.2 mM Na2
, 0.37 mM KH2
, 10 mM HEPES, and 7.5 mM glucose (500μl/well); cells were then incubated for 15 minutes at 37°C. The eNOS activity assay was initiated by incubating cells with PBS buffer (400 μl/well) containing 1.5 Ci/ml [3
-arginine for 15 minutes. The reaction was stopped by adding 1 N ice-cold TCA (500 μl/well). Cytosol preparations were transferred to ice-cold silanized glass tubes and extracted three times with water-saturated ether. The samples were neutralized with 1.5 ml of 25 mM HEPES (pH 8.0) and applied to Dowex AG50WX8 columns (Tris form) (Sigma Chemical Co., St. Louis, Missouri, USA). Columns were eluted with 1 ml of 40 mM HEPES buffer (pH 5.5) containing 2 mM EDTA and 2 mM EGTA. The eluate was collected in glass scintillation vials for [3
-citrulline quantitation by liquid scintillation spectroscopy. Activity of eNOS in cell lysates and tissue was also determined by a previously described immunoprecipitation assay (18
), using the columns described above. In brief, samples were split into two tubes, one for Western blotting and one for determination of eNOS activity. eNOS immunocomplexes immobilized on protein A–Sepharose beads were resuspended in assay buffer, and eNOS activity was determined by measuring the conversion of [3
-arginine into [3
-citrulline. Similar amounts of enzyme in each incubation were verified by eNOS blotting. All enzyme activities were corrected for [3
-arginine uptake into the cells under the various experimental conditions, determined using a previously described method (19
BAECs were incubated for 30 minutes in an uptake buffer (25 mM HEPES, 1.8 mM CaCl2, 5.4 mM KCl, 140 mM choline chloride, 0.8 mM MgSO4, and 5 mM glucose) containing 20 nM [3H]L-arginine. Uptake of L-arginine was terminated by adding ice-cold buffer, and cells were washed three times with 1 ml of buffer. After the final washing, cells were lysed by the addition of 1 ml 0.5% SDS in 0.1 N NaOH. Cellular lysates were added to 15 ml of Ecoscint-A scintillation fluid (Aquasol 2, Packard Instruments, Meriden, Connecticut, USA). The amount of [3H]L-arginine was determined by scintillation spectroscopy (LKB 1219 Rackbeta, PerkinElmer, Gaithersburg, Maryland, USA) and represented cellular transport of L-arginine.
Determination of mitochondrial membrane potential.
BAECs were cultured in 24-well plates (200,000 cells/well) and loaded with 0.5 μM JC-1 (Molecular Probes Inc., Eugene, Oregon, USA) in 20 mM HEPES and 0.1% BSA in MEM medium without phenol, according to the manufacturer’s instructions. Samples were viewed at room temperature with a cooled CCD camera and a high-resolution Olympus IX-70 microscope (Olympus America Inc., Melville, New York, USA) with 10× objectives. Epifluorescence optics with narrow-band FITC and rhodamine filters (Chroma Technology Corp., Brattleboro, Vermont, USA) were used to detect JC-1 green fluorescence and red fluorescence from JC-1 aggregates. Digital imaging was performed with a Photometrics PXL cold CCD camera (Roper Scientific Inc., Tucson, Arizona, USA) run by IPLab Spectrum software (Scanalytics Inc., Fairfax, Virginia, USA) on a Power Macintosh computer. Images were acquired randomly from selected fields.
Localization of recombinant MnSOD.
BAECs infected with MnSOD adenovirus were fixed with 2% paraformaldehyde for 10 minutes and postfixed for 2 minutes with methanol at –20°C. Cells were blocked with 20% horse serum/PBS for 30 minutes at room temperature. Cells were incubated with antimitochondrial serum and anti-MnSOD antibody for 1 hour at room temperature. Antigens were detected by immunofluorescence using anti-human IgG–Texas Red and anti-rabbit IgG-FITC conjugated secondary antibodies (Jackson ImmunoResearch Laboratories Inc., West Grove, Pennsylvania, USA). Confocal microscopic images were collected on a Radiance 2000 confocal microscope from Bio-Rad Laboratories Inc. (Hercules, California, USA) with a Kr/Ar laser, with excitation at 488 nm and 568 nm. Acousto optical tunable filter was attenuated so that there was no crosstalk from the green to red channel or vice versa. Images were taken in a single scan at 166 lines per second with a Nikon 60× NA 1.4 objective.
BAECs were plated in 100-mm cell culture plates and grown to confluence. Cells (2 × 107) were scraped from the plates, pelleted, and washed twice with cold PBS. The pellet was resuspended in cold lysis buffer (20 mM HEPES at pH 7.9, 500 mM NaCl, 20% glycerol, 1 mM DTT, 0.1% Nonidet P40, and 1 mM PMSF) and incubated on ice for 30 minutes with gentle vortexing. Cellular debris was pelleted for 20 minutes at 75,000 g at 4°C. The supernatants were dialyzed at 4°C overnight against binding buffer (20 mM HEPES at pH 7.9, 2 mM MgCl2, 10 μM ZnSO4, 1 mM DTT, 10% glycerol, and 1 mM PMSF).
Five hundred micrograms of protein was immunoprecipitated with 4 μg of the indicated antibody and 20 μl of protein A–Sepharose 4B (Amersham Pharmacia Biotech) in binding buffer (final concentration 1 μg protein/μl). The samples were rotated overnight at 4°C. The immunoprecipitated complexes were pelleted by centrifugation (1,000 g) and washed 4–5 times with binding buffer. The pellet was resuspended in 1× sample buffer, boiled, and analyzed by 7.5% SDS-PAGE with Western blotting. Aortae were homogenized in lysis buffer using a Dounce homogenizer, and were processed identically.
Immunoprecipitated proteins electrophoresed on 10% PAGE gels, and cytosolic proteins electrophoresed on 12% PAGE gels were transferred onto nitrocellulose membranes. The immunoblots were developed with 1:1,000 dilutions of the indicated antibodies, and the signal was detected with the ECL System according to the manufacturer’s instructions (Amersham Pharmacia Biotech). The images were scanned into a FluorImager (Molecular Dynamics, Sunnyvale, California, USA) and analyzed using the ImageQuant 5.5 program.
Sprague-Dawley rats weighing 200 grams were fasted overnight prior to streptozotocin injection. Animals were injected with 60 mg/kg of streptozotocin dissolved in sodium citrate buffer (pH 4.5). Seventy-two hours after injection, the animals were test-bled and serum glucose was determined. Mean serum glucose was 309 ± 8 for the diabetic group, and 103 ± 4 for the nondiabetic group. Animals were killed after 16 weeks of diabetes, and the aortae were dissected and washed free of blood.
Data were analyzed using the one–factor ANOVA procedure to compare the means of all groups. The Tukey-Kramer multiple-comparisons procedure was used to determine differences between means.