Angiotensin II increases the activity and synthesis of calcineurin in human neutrophils. (A) Neutrophils (6 × 106 cells/mL) were incubated with 10 nM Ang II for the indicated times, (B) with the indicated Ang II concentrations for 5 hours, or (inset) previously with 1 μg/mL CsA for 60 minutes and then with 10 nM Ang II for 5 hours. After cell lysis, calcineurin activity was assayed. Phosphatase activity is expressed as picomoles of phosphate released per minute per milligram protein. Mean ± SE values from 4 separate experiments performed in triplicate are presented. (C-D) Neutrophils (107 cells/mL) were previously incubated in the absence or presence of CsA for 1 hour or with 0.1 mM EGTA and 1 μM thapsigargin for 20 minutes, as indicated, and then were treated with 10 nM Ang II for another 5 hours. Proteins from lysed cells were resolved by SDS-PAGE, followed by immunoblotting with anticalcineurin. Both A (59-kDa) and B (19-kDa) subunits of calcineurin were detected (arrows). Thereafter, antibody stripping and reprobing of the membrane with antihuman β-actin was carried out.
