title: Regulation of intracellular pH mediates Bax activation in HeLa cells treated with staurosporine or tumor necrosis factor-alpha.
abstract: Induction of apoptosis in HeLa cells with staurosporine produced a rise in the intracellular pH (pH(i)). Intracellular alkalinization was accompanied by translocation of Bax to the mitochondria, cytochrome c release, and cell death. The chloride channel inhibitor furosemide prevented intracellular alkalinization, Bax translocation, cytochrome c release, and cell death. Translocation of full-length Bid to the mitochondria was also prevented by furosemide. The cleavage product of Bid degradation (truncated Bid, tBid) was not detectable in the mitochondria. Its accumulation in the cytosol was prevented by furosemide. Apoptosis induced by tumor necrosis factor-alpha (TNF) lowered pH(i), an effect also accompanied by Bax translocation, cytochrome c release, and cell killing. Furosemide prevented all of these events. TNF induced a depletion of full-length Bid from the mitochondria and the cytosol but induced an accumulation of mitochondrial tBid. Furosemide only delayed full-length Bid depletion and tBid accumulation. The caspase 8 inhibitor IETD did not prevent the translocation of Bax. Although IETD did inhibit the cleavage of Bid and the accumulation of tBid, cell killing was reduced only slightly. It is concluded that with either staurosporine or TNF a furosemide-sensitive change in pH(i) is linked to Bax translocation, cytochrome c release, and cell killing. With TNF Bax translocation occurs as Bid is depleted and can be dissociated from the accumulation of tBid. With staurosporine a role for full-length Bid in Bax translocation cannot be excluded but is not necessary as evidenced by the data with TNF.
authors.affiliation: Department of Pathology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
authors.forename: Marco
authors.initials: M
authors.lastname: Tafani
authors.forename: Joshua A
authors.initials: JA
authors.lastname: Cohn
authors.forename: Natalie O
authors.initials: NO
authors.lastname: Karpinich
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Rothman
authors.forename: Matteo A
authors.initials: MA
authors.lastname: Russo
authors.forename: John L
authors.initials: JL
authors.lastname: Farber
mesh_terms: D017209:Apoptosis; D051023:BH3 Interacting Domain Death Agonist Protein; D015153:Blotting, Western; D002138:Calibration; D002352:Carrier Proteins / Q000378:metabolism; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D002470:Cell Survival; D018118:Chloride Channels / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004232:Diuretics / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005665:Furosemide / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008928:Mitochondria / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D011485:Protein Binding; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D021381:Protein Transport; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2; D019311:Staurosporine / Q000494:pharmacology*; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D051028:bcl-2-Associated X Protein
chemical_list: C494826:BAX protein, human; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D002352:Carrier Proteins; D061945:Caspase Inhibitors; D018118:Chloride Channels; D003574:Cytochrome c Group; D004232:Diuretics; D004791:Enzyme Inhibitors; D009842:Oligopeptides; D011500:Protein Synthesis Inhibitors; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D014409:Tumor Necrosis Factor-alpha; D051028:bcl-2-Associated X Protein; C425644:isoleucyl-glutamyl-threonyl-aspartic acid fluoromethyl ketone; D005665:Furosemide; D003513:Cycloheximide; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D019311:Staurosporine