Figure 5.

Increased monomers and decreased zinc content in eNOS derived from BAECs exposed to high glucose are associated with increased O₂^.– and ONOO^–. (a) Increased eNOS monomers in cells exposed to high glucose. Confluent BAECs were exposed to control (5 mmol/l D-glucose) or high glucose (30 mmol/l) for 3 days. The eNOS protein was separated by low-temperature SDS-PAGE under reducing conditions, blotted, and detected with a monoclonal antibody. Blot shown is representative of results from eight independent experiments. (b) Increased eNOS monomer and correlated O₂^.– release are associated with decreased zinc content in eNOS from cells exposed to high glucose (n = 10, #P < 0.05). The zinc content of eNOS from cells incubated in elevated glucose was assayed as described in Methods and expressed as a percentage of that in control cells exposed to 5 mmol/l glucose. Of note, both L-NAME (1 mmol/l) and SOD-PEG (500 U/ml) prevented high glucose–induced dissociation of eNOS dimers (n = 8, *P < 0.01), zinc loss (n = 5, *P < 0.01), and O₂^.– release (n = 6, ^#P < 0.05 compared with cells exposed to high glucose alone). (c) eNOS-dependent tyrosine nitration of prostacyclin synthase (PGIS) in cells exposed to high glucose. PGIS was immunoprecipitated with a polyclonal antibody against PGIS (4 μg/ml). The nitrated PGIS was detected with a monoclonal antibody against 3-nitrotyrosine (3-NT) (1:1,000; Upstate Biotechnology Inc.). Of note, SOD-PEG (500 U/ml) or L-NAME (1 mmol/l), but not oxypurinol (10 μmol/l) or rotenone (10 μmol/l), prevented the nitration of PGIS. IP, immunoprecipitation; IB, immunoblot; PGIS, prostacyclin synthase. Blot shown is representative of three independent experiments.