title: The nitric oxide donor sodium nitroprusside requires the 18 kDa Translocator Protein to induce cell death.
abstract: Various studies have shown that several lethal agents induce cell death via the mitochondrial 18 kDa Translocator Protein (TSPO). In this study we tested the possibility that nitric oxide (NO) is the signaling component inducing the TSPO to initiate cell death process. Cell viability assays included Trypan blue uptake, propidium iodide uptake, lactate dehydrogenase release, and DNA fragmentation. These assays showed that application of the specific TSPO ligand PK 11195 reduced these parameters for the lethal effects of the NO donor sodium nitroprusside (SNP) by 41, 27, 40, and 42 %, respectively. TSPO silencing by siRNA also reduced the measured lethal effects of SNP by 50 % for all of these four assays. With 2,3-bis[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5-carboxyanilide (XTT) changes in metabolic activity were detected. PK 11195 and TSPO knockdown fully prevented the reductions in XTT signal otherwise induced by SNP. Collapse of the mitochondrial membrane potential was studied with the aid of JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine chloride). PK 11195 and TSPO knockdown reduced, respectively by 36 and 100 %, the incidence of collapse of the mitochondrial membrane potential otherwise induced by SNP. 10-N-Nonyl-Acridine Orange (NAO) was used to detect mitochondrial reactive oxygen species generation due to SNP. PK 11195 and TSPO knockdown reduced this effect of SNP by 65 and 100 %, respectively. SNP did not affect TSPO protein expression and binding characteristics, and also did not cause TSPO S-nitrosylation. However, β-actin and various other proteins (not further defined) were S-nitrosylated. In conclusion, TSPO is required for the lethal and metabolic effects of the NO donor SNP, but TSPO itself is not S-nitrosylated.
authors.affiliation: Department of Molecular Pharmacology, Faculty of Medicine, Rappaport Family Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology, Haifa, Israel.
authors.forename: Luba
authors.initials: L
authors.lastname: Shargorodsky
authors.forename: Leo
authors.initials: L
authors.lastname: Veenman
authors.forename: Beatriz
authors.initials: B
authors.lastname: Caballero
authors.forename: Yelena
authors.initials: Y
authors.lastname: Pe'er
authors.forename: Svetlana
authors.initials: S
authors.lastname: Leschiner
authors.forename: Julia
authors.initials: J
authors.lastname: Bode
authors.forename: Moshe
authors.initials: M
authors.lastname: Gavish
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D055785:Gene Knockdown Techniques; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D008024:Ligands; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D008970:Molecular Weight; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D009599:Nitroprusside / Q000494:pharmacology*; D015538:Nitrosation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D018079:Receptors, GABA / Q000378:metabolism*; D011963:Receptors, GABA-A / Q000378:metabolism*; D050994:Voltage-Dependent Anion Channels / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D007546:Isoquinolines; D008024:Ligands; D020030:Nitric Oxide Donors; D018079:Receptors, GABA; D011963:Receptors, GABA-A; C499229:TSPO protein, human; D050994:Voltage-Dependent Anion Channels; C069997:Tspo protein, rat; D009599:Nitroprusside; C037850:PK 11195