Long-term treatment with VEGF activates NO synthesis which nitrosylates NSF and inhibits exocytosis. (A) VEGF increases Akt phosphorylation. HAECs were pretreated with 50 ng/mL VEGF for 30 minutes, and total cell lysates were immunoblotted with antibody to phospho-Akt (top row) or total Akt (bottom row). (B) Dominant-negative Akt blocks VEGF inhibition of exocytosis. HAECs were transduced with adenovirus expressing dominant-negative Akt or adenovirus expressing β-galactosidase at an MOI of 200 for 48 hours, then treated with 50 ng/mL VEGF for 2 hours. Cells were then washed and treated with VEGF 50 ng/mL for 1 hour, and exocytosis was measured with an ELISA for VWF as described for Figure 1 (n = 3 ± SD; **P < .01 versus VEGF + LacZ). (C) Dominant-negative Akt inhibits VEGF-activated phosphorylation of eNOS. HAECs were transduced with adenoviral vectors expressing Akt(CA), Akt (DN), or LacZ at an MOI of 200 for 48 hours. Cells were then treated with 50 ng/mL VEGF for 30 minutes, and total cell lysates were immunoblotted with antibody to phospho-eNOS or eNOS.