Interactions among nitric oxide and Bcl‐family proteins after MPP⁺ exposure of SH‐SY5Y neural cells I: MPP⁺ increases mitochondrial NO and Bax protein

We studied effects of methylpyridinium ion (MPP + ) on apoptosis, cell death and regulation of Bcl‐2‐family proteins in SH‐SY5Y neuroblastoma cells. MPP + increased intracellular accumulation of DNA‐histone complexes as a measure of apoptosis and decreased intracellular calcein fluorescence as a measure of cell death. If ATP synthesis was supported, MPP + caused apoptosis in ρ 0 cells devoid of electron transport function. Caspase inhibition blocked apoptosis but not cell death caused by MPP + . MPP + increased levels of Bax, Bcl‐2 and Bcl‐X L proteins ∼2‐fold over 24 hr, with Bax increases occurring first; Bax did not increase in ρ 0 cells. The Bax increase, but not that of Bcl‐2 or Bcl‐X L , was dependent on nitric oxide (NO) and seemed post‐transcriptional. DAF‐FM imaging revealed increased mitochondrial NO within hours of exposure to MPP + . Western blots showed a constitutive ∼130 kD protein that stained for NOS‐2, consistent with reports of mitochondrial nitric oxide synthase (mtNOS). MPP + caused a NO‐dependent release of cytochrome C into cytoplasm. MPP + increases mitochondrial NO levels and causes a NO‐dependent increase in Bax protein, providing a mechanism for NOS‐and Bax‐dependency of MPTP neurotoxicity in vivo and implicating locally produced NO as a signaling molecule used by mitochondria to manipulate cell death cascades.