Nitric oxide prevents 6-hydroxydopamine-induced apoptosis in PC12 cells through cGMP-dependent PI3 kinase/Akt activation.
Nitric oxide (NO) functions not only as an important signaling molecule in the brain by producing cGMP, but also regulates neuronal cell apoptosis. The mechanism by which NO regulates apoptosis is unclear. In this study, we demonstrated that NO, produced either from the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) or by transfection of neuronal NO synthase, suppressed 6-hydroxydopamine (6-OHDA)-induced apoptosis in PC12 cells by inhibiting mitochondrial cytochrome c release, caspase-3 and -9 activation, and DNA fragmentation. This protection was significantly reversed by the soluble guanylyl cyclase inhibitor 1H-(1,2,4)-oxadiazole[4,3-a]quinoxalon-1-one, indicating that cGMP is a key mediator in NO-mediated anti-apoptosis. Moreover, the membrane-permeable cGMP analog 8-Br-cGMP inhibited 6-OHDA-induced apoptosis. These anti-apoptotic effects of SNAP and 8-Br-cGMP were suppressed by cGMP-dependent protein kinase G (PKG) inhibitor KT5823, indicating that PKG is a downstream signal mediator in the suppression of apoptosis by NO and cGMP. Both SNAP and 8-Br-cGMP induced endogenous Akt activation and Bad phosphorylation, resulting in the inhibition of Bad translocation to mitochondria; these effects were inhibited by KT5823 and the phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 and Wortmannin. Our data suggest that the NO/cGMP pathway suppresses 6-OHDA-induced PC12 cell apoptosis by suppressing the mitochondrial apoptosis signal via PKG/PI3K/Akt-dependent Bad phosphorylation.
Ha
Kwon-Soo
KS
Vascular System Research Center and Department of Molecular and Cellular Biochemistry, Kangwon National University, School of Medicine, Chunchon, Kangwon-do, Korea.
Kim
Ki-Mo
KM
Kwon
Young-Guen
YG
Bai
Se-Kyung
SK
Nam
Woo-Dong
WD
Yoo
Young-Min
YM
Kim
Peter K M
PK
Chung
Hun-Taeg
HT
Billiar
Timothy R
TR
Kim
Young-Myeong
YM
D000813:Anilides / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002454:Cell Differentiation; D006152:Cyclic GMP / Q000502:physiology*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism; D019610:Cytoprotection; D004789:Enzyme Activation; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D009569:Nitric Oxide / Q000502:physiology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000235:genetics; D052248:Nitric Oxide Synthase Type I; D009842:Oligopeptides / Q000378:metabolism; D016627:Oxidopamine / Q000037:antagonists & inhibitors*; D016716:PC12 Cells; D010396:Penicillamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D014162:Transfection
D000813:Anilides; D053148:Caspase 3; D020169:Caspases; D006152:Cyclic GMP; D017869:Cyclic GMP-Dependent Protein Kinases; D003574:Cytochrome c Group; D009569:Nitric Oxide; D020030:Nitric Oxide Donors; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D009842:Oligopeptides; D016627:Oxidopamine; D010396:Penicillamine; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; C110881:S-nitro-N-acetylpenicillamine; C430230:acetyl-aspartyl-glutamyl-valyl-aspartic acid p-nitroanilide; C496322:Nos1 protein, rat; C494920:Akt1 protein, rat; C505410:Casp3 protein, rat