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 rho(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 approximately 2-fold over 24 hr, with Bax increases occurring first; Bax did not increase in rho(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 approximately 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.
Dennis
Jameel
J
Neuroscience Graduate Program, University of Virginia School of Medicine, Charlottesville, Virginia 00908, USA.
Bennett
James P
JP
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D006801:Humans; D008928:Mitochondria / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D009447:Neuroblastoma / Q000378:metabolism; D009569:Nitric Oxide / Q000032:analysis / Q000096:biosynthesis*; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000096:biosynthesis*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis / Q000096:biosynthesis*; D011726:Pyridinium Compounds / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011726:Pyridinium Compounds; D051028:bcl-2-Associated X Protein; D009569:Nitric Oxide; C014677:1-methylpyridinium