Figure 2.

Endothelial nitric oxide synthesis controls PDI on the cell surface of endothelial cells. (A) HUVECs were incubated with either vehicle alone, 1 mM of L‐NAME, 100 μM histamine, or a combination of the latter two for 24 hours. NO levels were then evaluated using DAF‐FM fluorescence. (B) Thiol isomerase activity on the cell surface of HUVECs was assessed after incubation with either vehicle alone, 1 mM of L‐NAME, 100 μM histamine, or a combination of the latter two for 24 hours. (C) HUVECs incubated with either vehicle alone, 1 mM of L‐NAME, 100 μM histamine, or a combination of the latter two were assessed for S‐nitrosylation. Samples were lysed, free thiols were blocked with NEM and then acetone precipitated pellets were resuspended in the presence of 100 μM MPB and 5 mM ascorbic acid. After sample desalting, samples were loaded on streptavidin magnetic beads, washed and the elution was analyzed using SDS‐polyacrylamide gel electrophoresis and western blotting for PDI. Bar graphs represent RFU of DAF‐FM and di‐eosin‐GSSG, mean ± SEM (n = 3‐6). Statistical analysis for bar graphs was performed using one‐way analysis of variance with Bonferroni’s post test (*P < 0.05; **< 0.01). AUC, area under the curve; DAF‐FM, 4‐amino‐5‐methylamino‐2′,7′‐difluorofluorescein; GSSG, glutathione disulfide; l‐NAME, l‐N‐nitro‐l‐arginine methyl ester; PDI, protein disulfide isomerase; RFU, relative fluorescence units.