Phosphoinositide 3-kinase accelerates calpain-dependent proteolysis of fodrin during hypoxic cell death

We have shown recently that phosphoinositide 3-kinase (PI 3-kinase) accelerates the hypoxia-induced necrotic cell death of H9c2, derived from rat cardiomyocytes, by enhancing metabolic acidosis. Here we show the downstream events of acidosis that cause hypoxic cell death. Hypoxia induces the proteolysis of fodrin, a substrate of calpain. Intracellular Call chelation by BAPTA, and the addition of SJA6017, a specific peptide inhibitor of calpain, also reduces cell death and fodrin proteolysis, indicating that Ca2+ influx and calpain activation might be involved in these events. The overexpression of wild type PI 3-kinase accelerates fodrin proteolysis, while dominant-negative PI 3-kinase reduces it. Both (N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of the Na+/H+ exchanger, and KB-R7943, an inhibitor of the Na+/Ca2+ exchanger, reduce hypoxic cell death and fodrin proteolysis. The depletion of intracellular Call stores by thapsigargin,,an inhibitor of endoplasmic reticulum Ca2+-ATPase, also reduces cell death and fodrin proteolysis, indicating that Ca2+ release from intracellular Ca2+ stores might be also involved. These results indicate that PI 3-kinase might accelerate hypoxic cell death by enhancing the calpain-dependent proteolysis of fodrin.