OXIDANT INJURY IN PC12 CELLS - A POSSIBLE MODEL OF CALCIUM DYSREGULATION IN AGING .1. SELECTIVITY OF PROTECTION AGAINST OXIDATIVE STRESS

Previous research has suggested that the initial effects of cellular f ree radical neurotoxic insult involve large increases in intracellular Ca2+. However, the exact role of oxidative stress on the Various para meters involved in these increases has not been specified. The present experiments were performed to examine these parameters in PC12 cells exposed to 5, 25, or 300 mu M H2O2 for 30 min in growth medium alone o r containing either nifedipine (L-type Ca2+ antagonist), conotoxin (N- type antagonist), Trolox (vitamin E analogue), or alpha-phenyl-n-tert- butylnitrone (nitrone trapping agent; PEN). The concentrations of H2O2 were chosen by examining the degree of cell killing induced by exposu re to graded concentrations of H2O2. The 5 and 25 mu M concentrations of H2O2 produced no significant cell killing at either 30 min or 24 h after treatment, whereas the 300 mu M concentration produced a moderat e degree of cell killing that did not increase between the two times. Fluorescent imaging was used to visualize intracellular Ca2+ changes i n fura-2-loaded cells. Baseline (pre-30 mM KCI) Ca2+ levels were incre ased significantly by H2O2 treatment (e.g., 300 mu M, 200%), but the r ise in the level of free intracellular Ca2+ after KCI stimulation (i.e ., peak) was decreased (e.g., 300 mu M, 50%) and the cell's ability to sequester or extrude the excess Ca2+ (i.e., Ca2+ recovery time) after depolarization was decreased significantly, All compounds prevented b aseline Ca2+ increases and, with the exception of conotoxin, antagoniz ed the peak decreases in Ca2+. It is interesting that after 300 mu M H 2O2 exposure, only Trolox was partially effective in preventing these deficits in recovery. Conotoxin increased the decrement recovery in th e absence of H2O2. However, in cells exposed to 5 or 25 mu M H2O2, con otoxin as well as the other agents were effective in preventing the de ficits in recovery.