Regulation of CA-4-P–induced membrane blebbing.

Confluent endothelial cultures grown on Permanox slides were pretreated with either C3 exoenzyme (24 hours, 10 μg/mL) or for 30 minutes with all other specified inhibitors/activators used either singly or in combination at the following concentrations: Y-27632, 10 μM; HA1077, 10 μM; cytochalasin D, 10 nM; SB203580, 10 μM; PD98059, 25 μM; PDBu, 200 nM; ML-7, 10 μM; ML-9, 10 μM; and BDM, 5 mM. Cells were then exposed to CA-4-P (60 minutes, 1 μM), stained with Texas Red–conjugated phalloidin, and mounted in Vectashield with DAPI. Blebbing was quantified by counting phalloidin-stained blebbing cells in 9 random microscope fields per treatment and expressing these as a proportion of total numbers of DAPI-stained nuclei in identical fields. Values are means ± SD from 4 independent experiments using HUVEC cultures at passages 2 to 3. *P < .05 compared with vehicle + CA-4-P.