ATP-induced activation of A-SMase results in increased hydrolysis of SM on the cell surface. (A) MDMs were metabolically labeled with [methyl-¹⁴C]-choline chloride (0.2 μCi/mL) for 48 hours. The labeled cells were treated with a control vehicle (60 minutes) or BzATP (200 µM) for varying times and supernatants were collected. The cell supernatants were subjected to centrifugation at 400g for 5 minutes and then 21 000g for 60 minutes to remove cell debris and pellet MVs, respectively. The supernatants devoid of cell debris and MVs were counted for the radioactivity to determine the release of [¹⁴C]-phosphorylcholine. (B) MDMs treated with a control vehicle or BzATP (200 µM) for 10 minutes were analyzed for their ability to bind lysenin, a protein that specifically binds SM, as described in the legend to Figure 2. (C) The quantification of lysenin binding to cells. The fluorescence intensity of cells (30 cells or more) was measured using ImageJ2 software. (D) MDMs metabolically labeled with [methyl-¹⁴C]-choline chloride as described previously were treated with A-SMase inhibitors desipramine or imipramine for 1 hour and then stimulated with BzATP (200 µM) for 1 hour. The released radioactivity ([¹⁴C]-phosphorylcholine) was measured to monitor the hydrolysis of SM in the outer leaflet. (E) MDMs transfected with mock, scRNA, or siRNA specific for A-SMase were metabolically labeled with [methyl-¹⁴C]-choline chloride (0.2 μCi/mL) for 48 hours. The cells were then stimulated with a control vehicle or ATP for 60 minutes, and the release of radioactivity ([¹⁴C]-phosphorylcholine) was measured. Mann-Whitney U test was used to determine statistical significance between 2 groups. *P < .05; **P < .01; *** P < .001.