Effect of t-butyl hydroperoxide on Ca ²⁺ movement in PC12 pheochromocytoma cells

The effect of the oxidant t-butyl hydroperoxide on intracellular free levels of Ca ²⁺ ([Ca ²⁺ ]i) in PC12 pheochromocytoma cells was examined by using fura-2 as a fluorescent dye. t-Butyl hydroperoxide induced an increase in [Ca ²⁺ ] _i in a concentration-dependent fashion between 50-250 μM with an EC50 of 100 μM. The [Ca ²⁺ ] _i signal consisted of a slow rise and a sustained phase. The response was decreased by 65% by removal of extracellular Ca ²⁺ . In Ca ²⁺ -free medium, pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca ²⁺ pump inhibitor) abolished 150 μM t-butyl hydroperoxide-induced [Ca ²⁺ ] _i increase, and conversely, pretreatment with t-butyl hydroperoxide abrogated thapsigargin-induced [Ca ²⁺ ] _i increase. The 150 μM t-butyl hydroperoxide-induced [Ca ²⁺ ] _i increase in Ca ²⁺ medium was reduced by 42±5% by pretreatment with 0.1 μM nicardipine but not by 10 μM verapamil, nifedipine, nimodipine or diltiazem, or by 50 μM La ³⁺ or Ni ²⁺ . Pretreatment with 10 μM t-butyl hydroperoxide for 40 min did not affect 10 μM ATP-induced [Ca ²⁺ ] _i increase. Together, the results show that t-butyl hydroperoxide induced significant [Ca ²⁺ ] _i increase in PC12 cells by causing store Ca ²⁺ release from the thapsigargin-sensitive endoplasmic reticulum pool in an inositol 1,4,5-trisphosphate-independent manner and by inducing Ca ²⁺ influx via a nicardipine-sensitive pathway.