Figure 3
The Ca2+ ionophore A23187 increases MAO-A activity. (A) Levels of free intracellular Ca2+, [Ca2+]i, in HT-22 cells treated with A23187 (5 μM: 30 min) were determined using the Ca2+-binding Fluo-3 AM fluorescent dye. (B) MAO-A and MAO-B activities were assessed radioenzymatically in A23187-treated cell cultures (*P < 0.05 versus control levels). (C) MAO-A protein expression was determined in SDS-PAGE resolved total cell lysates. Levels of β-actin demonstrate equal protein loading. (D) Densitometry graph representing mao-A gene expression (as a ratio to GAPDH expression) determined using semi-quantitative RT-PCR amplification of mRNA extracted from A23187-treated cell cultures. (E) Representative RT-PCR amplification fragments. (F) In similarly-treated cells, the production of ROS was assessed using the H2O2-binding DCF fluorogen. A parallel series of cell cultures were pre-treated with the specific MAO-A inhibitor, clorgyline (CLG; 100 μM, 1 h). Data represent mean ± SD.