PI-PLC activity involvement in Dex-induced thymocyte apoptosis. Effect of U73122 on (A) Dex-induced PI-PLC activity, (B) aSMase (left) and nSMase (right), (C) ceramide generation, and (D) apoptosis. Thymocytes were treated with Dex (10−7 mol/L) for the indicated times in the presence or absence of U73122 (2.5 μmol/L) or D609 (50 μg/mL). (A) Dex-induced PI-PLC activity. Cell extracts were reacted with radiolabeled PI vesicles, and then DAG released was separated by TLC, visualized by autoradiography, scraped from the plate, and counted by scintillation counting. PI-PLC activity is expressed as picomoles DAG/106 cells (left side). Mean values of three different experiments in duplicate are reported. Standard deviations (SD), less than 3% of the mean values, are omitted for clarity. On the right side, a representative autoradiography of a DAG chromatogram is shown. (B) Acidic (on the left) and neutral (on the right) SMase activity induced after 15 or 120 minutes of Dex treatment, respectively. Hydrolyzed SM was quantitated and expressed as picomoles/106 cells. Mean values of three different experiments in duplicate are reported. SD, less than 5% of the mean values, are omitted for clarity. (C) Ceramide levels in thymocytes treated for 15 minutes with Dex (10−7 mol/L) in the presence or absence of U73122. The quantitative results for ceramide-1-phosphate levels, expressed as picomoles/106cells, are reported (mean values from three experiments in duplicate; SD values, less than 10% of mean values, are omitted for clarity). (D) Effect of U73122 and D609 on Dex-induced thymocyte apoptosis, as detected after an 18-hour culture. Percentage numbers of hypodiploid nuclei are reported for each condition. The results are representative of one out of three separate experiments.
