Figure 6.

Effects of BAs on Ca²⁺ release, MAPK activation, and ROS production in granulocytic HL60 cells. HL60 cells were differentiated towards granulocytic cells in the presence of 1.5% DMSO, 2 ng ml⁻¹ TGFβ, and 500 pM calcitriol for 4 days. (a) Mobilisation of Ca²⁺. Cells (10⁷ ml⁻¹ PGC buffer) were loaded with 2 μM Fura-2 for 30 min at 37°C. After addition of BAs (50 μM, each) or 1 μM fMLP, the fluorescence was measured and intracellular free Ca²⁺ was calculated as described. The monitored curves show one typical experiment out of 4–5, respectively. (b) MAPK activation. Cells (5 × 106/100 μl PGC buffer) were stimulated with 30 μM of BAs, 1 μM fMLP, or left untreated. After 1.5 min at 37°C, incubations were terminated by addition of the same volume of ice-cold SDS-b. Samples were electrophoresed and analysed for dually phosphorylated p38 MAPK or p44/42^MAPK by Western blotting. (c) ROS formation. Cells (5 × 10⁷ ml⁻¹ PGC buffer) were preincubated with DCF-DA (10 μg ml⁻¹) for 2 min at 37°C prior addition of the indicated stimuli. The generation of peroxides was measured as described. Data determined 5 min after addition of stimuli are expressed as the mean of the fluorescence given in arbitrary units±s.e., n=4. Student's t-test; ^*P<0.05; ^**P<0.01.