Role of calcium in 5-LO product formation, 5-LO translocation, and p38 MAPK activation.
(A) Ca++ influx. To Fura-2–loaded PMNLs (1 × 107/ mL PGC buffer) the indicated stimuli were added and the fluorescence was measured. Intracellular free Ca++ was calculated as described. The monitored curves show one typical experiment of 3 to 4. (B) 5-LO product formation. CaCl2 (1 mM), EDTA (1 mM), and BAPTA/am (30 μM) were added to 5 × 106 freshly isolated PMNLs in PG buffer as indicated. After 10 minutes at 37°C, SA (10 μM) and NaCl (0.3 M) were added 3 minutes prior to addition of 40 μM AA. Ionophore A23187 (2.5 μM), thapsigargin (1 μM), or fMLP (1 μM) was added simultaneously with AA. After another 5 minutes, 5-LO products were determined. Results are given as mean + SE (n = 3). Student t test; *P < .05; **P < .01. (C) 5-LO translocation. Freshly isolated PMNLs (3 × 107 in 1 mL PG buffer) were preincubated for 10 minutes at 37°C with 1 mM CaCl2 or with 1 mM EDTA and 30 μM BAPTA/am as indicated. Then, 100 μM SA or 2.5 μM ionophore was added to the samples and incubated for another 5 minutes at 37°C. 5-LO was detected in nuclear fractions by immunoblotting after subcellular fractionation. Similar results were obtained in 2 additional independent experiments. (D) Activation of p38 MAPK. Freshly isolated PMNL (5 × 106 in 100 μL PG buffer) were treated with 1 mM CaCl2 or with 1 mM EDTA and 30 μM BAPTA/am as indicated for 10 minutes at 37°C. Ionophore (2.5 μM), SA (10 μM), or NaCl (0.3 M) was then added and after 3 minutes at 37°C samples were analyzed for dually phosphorylated p38 MAPK. Results are representative of at least 3 separate experiments.
