The specific Na+/Ca2+ exchange inhibitor SEA0400 prevents nitric oxide-induced cytotoxicity in SH-SY5Y cells

The Na+/Ca2+ exchanger (NCX) plays a role in the regulation of intracellular Ca2+ levels, and nitric oxide (NO) is involved in many pathological conditions including neurodegenerative disorders. We have previously found that sodium nitroprusside (SNP), an NO donor, causes apoptotic-like cell death in cultured glial cells via NCX-mediated pathways and the mechanism for NO-induced cytotoxicity is cell type-dependent. The present study examined using the specific NCX inhibitor 2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline (SEA0400) whether NCX is involved in NO-induced injury in cultured neuronal cells. The treatment of neuroblastoma SH-SY5Y cells with SNP resulted in apoptosis and the cytotoxicity was blocked by the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase inhibitor U0126 and the p38 MAP kinase (MAPK) inhibitor SB203580, but not by the c-Jun N-terminal kinase (JNK) inhibitor SP60012. SNP increased Ca2+ influx and intracellular Ca2+ levels. In addition, SNP increased ERK and p38 MAPK phosphorylation, and production of reactive oxygen species (ROS) in an extracellular Ca2+-dependent manner. These effects of SNP were prevented by SEA0400. SNP-induced cytotoxicity was not affected by inhibitors of the Ca2+, Na+ and store-operated/capacitative channels. Moreover, SNP-induced increase in intracellular Ca2+ levels, ROS production and decrease in cell viability were blocked by a cGMP-dependent protein kinase (PKG) inhibitor. These results suggest that Ca2+ influx via the reverse of NCX is involved in the cascade of NO-induced neuronal apoptosis and NO activates the NCX through guanylate cyclase/PKG pathway.

► SNP-induced cytotoxicity was blocked by MAPK inhibitors in neuroblastoma cells.
► The effects of SNP were dependent on extracellular Ca2+.
► SNP increased Ca2+ influx and intracellular Ca2+ levels.
► These effects of SNP were prevented by the specific NCX inhibitor SEA0400.
► NCX-mediated Ca2+ influx plays a key role in NO-induced neuronal apoptosis.