PKA activation and Stx2B-induced VWF secretion. (A) HUVECs in buffer containing 0.75mM IBMX were treated for the indicated times without (IBMX, ▴) or with 20μM forskolin (▵), 5nM Stx1 (●), 5nM Stx1B (○), 5nM Stx2 (■), or 5nM Stx2B (□), and intracellular cAMP concentration was measured using a CatchPoint cAMP fluorescent assay kit. (B) HUVECs expressing CFP-Epac-YFP were examined by fluorescence microscopy, CFP and YFP fluorescence emission was monitored, and 5nM Stx1B (○) or Stx2B (□) was added at 50 seconds. As a positive control, forskolin (20μM) was added, and cells were monitored another 200 seconds. The CFP/YFP fluorescence intensity ratio reflects the intracellular cAMP level. (C) HUVECs in parallel plate perfusion chambers were pretreated without or with the PKA inhibitors 5μM H89 or 1mM 6-22 amide, followed by 5nM Stx1B or Stx2B. Control cells were perfused with buffer only. VWF strings were counted and values normalized to the Stx1B condition. H89 and 6-22 amide inhibited the secretion of VWF induced by Stx2B (P < .0001) but not by Stx1B. (D) HUVECs were treated 5 minutes without (Control) or with 20μM forskolin, 5nM Stx1B, or 5nM Stx2B. Cell lysates were assayed for PKA activity with a synthetic peptide substrate (top) or analyzed by SDS-PAGE and Western blotting for phosphorylated 14-3-3ζ or GAPDH (bottom). (E) HUVECs were transfected with control siRNA (Ctrl) or PKA siRNA. After 72 hours, cell lysates (top) were analyzed by SDS-PAGE and Western blotting for PKA or tubulin. Parallel cultures treated similarly (bottom) were perfused with 5nM Stx1B or Stx2B. VWF strings were counted and normalized to the Stx1B control condition. *P < .0005, NS indicates P = .25 by Student t test.