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Expression of an endothelial-type nitric oxide synthase isoform in human neutrophils: modification by tumor necrosis factor-alpha and during acute myocardial infarction

^a Cardiovascular Research and Hypertension Laboratory, Fundación Jiménez Díaz, Madrid, Spain

View larger version (7K): [in a new window]   Figure 1 Endothelial-type NOS RNA probes used in gel mobility shift assays. The diagram shows the different fragments of the 3'-UTR from eNOS mRNA used in this study. The entire eNOS mRNA is not drawn to scale. 3'-UTR = 3'-untranslated region; eNOS = endothelial-type nitric oxide synthase; mRNA = messenger ribonucleic acid; nt = nucleotide.

View larger version (11K): [in a new window]   Figure 2 Representative Western blot analysis demonstrating the expression of eNOS protein in TNF-alpha–incubated (500 U/ml) human neutrophils. Human neutrophils were incubated for different periods with TNF-alpha (500 U/ml). (Densitometric analysis in arbitrary units—0 h: 100; 2 h: 117 ± 8; 4 h: 108 ± 9; 6 h: 60 ± 7*; 8 h: 23 ± 8*; [n = 6];*p < 0.05 vs. 0 h.) eNOS = endothelial-type nitric oxide synthase.

View larger version (9K): [in a new window]   Figure 3 A, Representative Northern blot analysis of eNOS mRNA expression after exposure of human neutrophils to TNF-alpha (500 U/ml) for different periods. The experiments were performed in the presence of the transcriptional inhibitor DRB (60 µmol/liter). B, The graph shows the densitometric value of the eNOS hybridization signal relative to glyceraldehyde-3-phosphate dehydrogenase, expressed as a percentage of the control value in the absence of TNF-alpha. The GAPDH probe was used as a loading control for loading. *p < 0.05 vs. 0 h. DRB = 5,6-dichloro-1-2-D-ribofuranosylbenzimidazole; eNOS = endothelial-type nitric oxide synthase; GAPDH = glyceraldehyde-3-phosphate dehydrogenase.

View larger version (17K): [in a new window]   Figure 4 A, Representative gel mobility shift assay of six different experiments with the 3'-UTR of eNOS mRNA. The autoradiograph shows the results of gel mobility shift assays using the labeled UTR-L probe incubated with neutrophil cytosolic extracts (lane 1). Competitive studies were performed with 1,000 ng of unlabeled UTR-L (lane 2). Two RNA probes containing specific sequences within UTR-L were also tested as competitors: 1,000 ng of UTR-UC (lane 3) and 1,000 ng of UTR-AU (lane 4). The amount of the labeled UTR-L used was 1 ng. B, Treatment of neutrophil cytosolic extracts with proteinase K (87 µg/ml) before incubation with UTR-L RNA fully abolished the gel-shifted band. Abbreviations as in Figure 1.

View larger version (25K): [in a new window]   Figure 5 A, Representative gel mobility shift assay of six different experiments to analyze the regulation of neutrophil cytosolic protein binding activity by TNF-alpha. Human neutrophils were incubated with TNF-alpha (500 U/ml) for different periods, and their cytosolic extracts were analyzed by gel mobility shift assay using the labeled UTR-UC probe. B, Competitive experiments with greater amounts of unlabeled UTR-UC probe, demonstrating the specificity of the binding. For the competition experiments, human neutrophils were incubated with TNF-alpha (500 U/ml) for 8 h. The amount of radiolabeled RNA used was 1 ng. 1x = 10 ng of RNA.

View larger version (60K): [in a new window]   Figure 6 Representative gel mobility shift assay of six different experiments to test the effect of simvastatin on the binding activity of human neutrophil cytosolic proteins to the 3'-UTR of eNOS mRNA. The neutrophils were stimulated with TNF-alpha (500 U/ml) for 8 h in the presence and absence of increasing doses of simvastatin. The cytosolic extracts were analyzed using the labeled UTR-UC probe. (Densitometric analysis in arbitrary units—basal: 100; TNF-alpha: 180 ± 10; TNF-alpha + simvastatin 10–7 mol/liter: 142 ± 17*; TNF-alpha + simvastatin 10–6 mol/liter: 120 ± 9*; TNF-alpha + simvastatin 10–5 mol/liter: 45 ± 7*; [n = 6];*p < 0.05 vs. TNF-alpha–incubated neutrophils.) Abbreviations as in Figure 1.

View larger version (10K): [in a new window]   Figure 7 Representative Western blot analysis demonstrating the effect of simvastatin (10–5 mol/liter) on eNOS expression in TNF-alpha–stimulated (500 U/ml) human neutrophils. Expression of eNOS was determined 8 h after TNF-alpha stimulation. (Densitometric analysis in arbitrary units—basal: 100*; TNF-alpha: 23 ± 8; TNF-alpha + simvastatin 10–5 mol/liter: 211 ± 19*; [n = 6]; *p < 0.05 vs. TNF-alpha–incubated neutrophils.) eNOS = endothelial-type nitric oxide synthase.

View larger version (24K): [in a new window]   Figure 8 A, Representative Western blot analysis showing eNOS protein expression in neutrophils obtained from a patient with AMI and from a control subject. (Densitometric analysis in arbitrary units—control subject: 100; patient with AMI: 16 ± 4 [n = 8]; *p < 0.05.) B, Representative gel mobility shift assay to analyze the presence of the cytosolic proteins that bind to 3'-UTR of eNOS mRNA in neutrophils from both a control subject and a representative patient with AMI. Competitive experiments were performed with 1,000 ng of unlabeled UTR-UC demonstrating the specificity of the binding. Treatment of the neutrophil cytosolic extracts from patients with AMI with proteinase K (Prot K, 87 µg/ml) before incubation with the UTR-L RNA fully abolished the gel-shifted band. 3'-UTR = 3'-untranslated region; AMI = acute myocardial infarction.