Does L-NAME Directly Activate Mast Cells?
Since L-NAME-treated mast cells were able to induce neutrophil adhesion even if endothelium was removed from the system, we initially examined whether L-NAME was directly affecting the mast cells. First, oxidative stress was measured in mast cells. A stock solution of DHR 123 was diluted in PBS just prior to study. Mast cells were resuspended in HTB at a concentration of 4 times 105 cells/mL and preloaded with DHR 123 (5 micro mol/L) at 37 degrees C for 20 minutes, with gentle mixing every 5 minutes. After incubation, the mast cells were washed with cold HTB (800 rpm for 4 minutes) and resuspended in HTB. Aliquots of cells were then removed and incubated with L-NAME, L-NAME plus L-arginine, or L-NAME plus various antioxidants, including SOD (60 micro gram/mL), butylated hydroxytoluene (0.1 mmol/L), alpha, alpha prime-dipyridyl (2 mmol/L), and the iron chelator desferrioxamine (0.2 mmol/L), for 30 minutes at 37 degrees C. Since mitochondria are known to convert 1% to 4% of all oxygen to
