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Figure 1. Stimulation of eNOS increases protein glutathiolation. A) Vascular tone of isolated rings prepared from rat aorta precontracted by phenylephrine (PE, 1 µM) and stimulated by ACh (ACh, 1 µM) in the absence or the presence of 100 µM L-NAME. Control rings were equilibrated in buffer alone. B) Photomicrographs of untreated rings (Control) or rings treated with ACh or ACh + L-NAME). Rings were fixed and stained with the anti-PSSG Ab. C) Relative intensity of anti-PSSG Ab staining in rings treated with ACh ± L-NAME. Staining intensity was quantified using Metamorph software and normalized to that of buffer-incubated rings. D) Intensity of immunoblots from homogenates of aortic rings that were either left untreated (Control) or treated with ACh ± L-NAME. Glutathiolated proteins were detected by immunoblotting with anti-PSSG antibodies. Inset shows images of blots developed using anti-PSSG Ab after treatment with ß-mercaptoethanol (BME). E) Two-dimensional Western blots of aortic rings precontracted with PE and relaxed with ACh in the absence (a) or presence (b) of L-NAME. Whole tissue extracts were subjected to 2D electrophoresis, and glutathiolated proteins were detected by anti-PSSG antibodies. a, Inset) Same blot after stripping and reprobing for smooth muscle actin. Proteins 1–6 were excised from corresponding Sypro Ruby-stained gels and identified by MALDI-TOF/MS. Data are representative of 4 individual experiments. Bars are mean ± SE; *P < 0.05 vs. control; **P < 0.01 vs. ACh.
