FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1963564407> ?p ?o ?g. }

• W1963564407 endingPage "49576" @default.
• W1963564407 startingPage "49569" @default.
• W1963564407 abstract "Induction of apoptosis in HeLa cells with staurosporine produced a rise in the intracellular pH (pHi). Intracellular alkalinization was accompanied by translocation of Bax to the mitochondria, cytochromec 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-α (TNF) lowered pHi, an effect also accompanied by Bax translocation, cytochrome crelease, 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 pHi 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. Induction of apoptosis in HeLa cells with staurosporine produced a rise in the intracellular pH (pHi). Intracellular alkalinization was accompanied by translocation of Bax to the mitochondria, cytochromec 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-α (TNF) lowered pHi, an effect also accompanied by Bax translocation, cytochrome crelease, 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 pHi 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. Bax is a proapoptotic member of the Bcl-2 family of proteins that is implicated in the pathogenesis of cell death in an increasing number of models of apoptosis both in vivo and in vitro. In particular, Bax has emerged as a mediator of the mitochondrial phase of apoptosis, a process that culminates in the release of cytochromec from the intermitochondrial space and the activation of effector caspases. Bax is constitutively present in many cell types that undergo apoptosis in response to a variety of stimuli. By contrast, in other cells Bax expression is induced by activation of p53 upon damage to the genome or interference with the normal progression of the cell cycle (reviewed in Ref. 1Wu X. Deng Y. Front. Biosci. 2002; 7: 151-156Crossref PubMed Google Scholar). Whether constitutively expressed or induced, however, the primary action of Bax is a consequence of its translocation from the cytosol to the mitochondria. Clearly, where Bax is constitutively present in the cytosol, a mechanism that is referred to as Bax activation follows the introduction of an apoptotic stimulus to cause translocation of the protein to the mitochondria. Translocation of preformed Bax from the cytosol to the mitochondria has been reported with a variety of apoptotic stimuli (2Hsu Y.T. Wolter K.G. Youle R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3668-3672Crossref PubMed Scopus (1021) Google Scholar, 3Wolter K.G. Hsu Y.T. Smith C.L. Nechushtan A. Xi X.G. Youle R.J. J. Cell Biol. 1997; 5: 1281-1292Crossref Scopus (1562) Google Scholar, 4Hsu Y.T. Youle R.J. J. Biol. Chem. 1998; 273: 10777-10783Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar, 5Gross A. Jockel J. Wei M.C. Korsmeyer S.J. EMBO J. 1998; 17: 3878-3885Crossref PubMed Scopus (961) Google Scholar, 6Desagher S. Osen-Sand A. Nichols A. Eskes R. Montessuit S. Lauper S. Maundrell K. Antonsson B. Martinou J.-C. J. Cell Biol. 1999; 144: 891-901Crossref PubMed Scopus (1089) Google Scholar, 7Eskes R. Desagher S. Antonsson B. Martinou J.-C. Mol. Cell. Biol. 2000; 20: 929-935Crossref PubMed Scopus (1010) Google Scholar, 8Murphy K.M. Ranganathan V. Farnsworth M.L. Kavallaris M. Lock R.B. Cell Death Differ. 2000; 7: 102-111Crossref PubMed Scopus (285) Google Scholar, 9Deng Y. Wu X. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12050-12055Crossref PubMed Scopus (175) Google Scholar, 10Tsujimoto Y. Shimizu S. FEBS Lett. 2000; 466: 6-10Crossref PubMed Scopus (628) Google Scholar, 11Tafani M. Minchenko D.A. Serroni A. Farber J.L. Cancer Res. 2001; 61: 2459-2466PubMed Google Scholar). In the situation where Bax is synthesized upon introduction of an apoptotic stimulus, evidence exists that a similar mechanism of Bax activation is operative to control its translocation to the mitochondria (12Karpinich N.O. Tafani M. Rothman R.J. Russo M.A. Farber J.L. J. Biol. Chem. 2002; 277: 16547-16552Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). In the absence of an activating signal, Bax translocation is likely prevented by the interaction of the C-terminal segment of the protein with its N-terminal domain, an effect that prevents insertion of the hydrophobic C terminus into the mitochondria (2Hsu Y.T. Wolter K.G. Youle R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3668-3672Crossref PubMed Scopus (1021) Google Scholar, 13Goping I.S. Gross A. Lavoie J.N. Nguyen M. Jemmerson R. Roth K. Korsmeyer S.J. Shore G.C. J. Cell Biol. 1998; 143: 207-215Crossref PubMed Scopus (544) Google Scholar, 14Nechushtan A. Smith C.L. Hsu Y.T. Youle R.J. EMBO J. 1999; 18: 2330-2341Crossref PubMed Scopus (622) Google Scholar, 15Khaled A.R. Kim K. Hofmeister R. Muegge K. Durum S.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14476-14481Crossref PubMed Scopus (217) Google Scholar, 16Pawlowski J. Kraft A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 529-531Crossref PubMed Scopus (171) Google Scholar). In fact, deletion of the N terminus of Bax resulted in mitochondrial localization of the molecule in the absence of an apoptotic stimulus (13Goping I.S. Gross A. Lavoie J.N. Nguyen M. Jemmerson R. Roth K. Korsmeyer S.J. Shore G.C. J. Cell Biol. 1998; 143: 207-215Crossref PubMed Scopus (544) Google Scholar, 14Nechushtan A. Smith C.L. Hsu Y.T. Youle R.J. EMBO J. 1999; 18: 2330-2341Crossref PubMed Scopus (622) Google Scholar). At least two models have been proposed to account for the mechanism of Bax activation in apoptosis. An interaction of Bax with another proapoptotic protein may trigger Bax activation. In particular, the proapoptotic protein Bid induced a change in Bax conformation that resulted in exposure of its N-terminal domain and its integration into mitochondrial membranes (6Desagher S. Osen-Sand A. Nichols A. Eskes R. Montessuit S. Lauper S. Maundrell K. Antonsson B. Martinou J.-C. J. Cell Biol. 1999; 144: 891-901Crossref PubMed Scopus (1089) Google Scholar, 7Eskes R. Desagher S. Antonsson B. Martinou J.-C. Mol. Cell. Biol. 2000; 20: 929-935Crossref PubMed Scopus (1010) Google Scholar). The evidence that such a Bid-dependent mechanism of Bax activation operates in an intact cell is based largely on staurosporine-induced apoptosis (6Desagher S. Osen-Sand A. Nichols A. Eskes R. Montessuit S. Lauper S. Maundrell K. Antonsson B. Martinou J.-C. J. Cell Biol. 1999; 144: 891-901Crossref PubMed Scopus (1089) Google Scholar). Mice deficient in Bid, however, were not resistant to the cell killing by staurosporine (17Yin X.-M. Wang K. Gross A. Zhao Y. Zinkel S. Klocke B. Roth K.A. Korsemeyer S.J. Nature. 1999; 400: 886-891Crossref PubMed Scopus (860) Google Scholar). Another concern with the Bid-dependent model is that the problem of Bax activation simply becomes the problem of Bid activation. Like Bax cytosolic Bid exists in an inactive conformation (18Chou J.J. Li H. Salvesen G.S. Yuan J. Wagner G. Cell. 1999; 96: 615-624Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar, 19McDonnell J.M. Fushman D. Milliman C.L. Korsmeyer S.J. Cowburn D. Cell. 1999; 96: 625-634Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). Caspase-8 cleaved Bid to generate an active truncated Bid (tBid) 1The abbreviations used are: tBid, truncated Bid; TNF, tumor necrosis factor-α; IL, interleukin; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; HBSS, Hanks' balanced salt solution; BCECF-AM, 2′,7′-bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein), acetoxymethylester; CHX, cycloheximide; STR, staurosporine; pHi, intracellular pH; PIPES, 1,4-piperazinediethanesulfonic acid1The abbreviations used are: tBid, truncated Bid; TNF, tumor necrosis factor-α; IL, interleukin; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; HBSS, Hanks' balanced salt solution; BCECF-AM, 2′,7′-bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein), acetoxymethylester; CHX, cycloheximide; STR, staurosporine; pHi, intracellular pH; PIPES, 1,4-piperazinediethanesulfonic acid that induced the release of cytochrome c from mitochondria (20Luo X. Budharji I. Zou H. Slaughter C. Wang X. Cell. 1988; 94: 481-490Abstract Full Text Full Text PDF Scopus (3050) Google Scholar, 21Li H. Zhu H.Xu, C.-J. Yuan J. Cell. 1998; 94: 491-501Abstract Full Text Full Text PDF PubMed Scopus (3756) Google Scholar, 22Gross A. Yin X.-M. Wang K. Wei M.C. Jockel J. Milliman C. Erdjument-Bromage H. Tempst P. Korsmeyer S.J. J. Biol. Chem. 1999; 274: 1156-1163Abstract Full Text Full Text PDF PubMed Scopus (922) Google Scholar). Engagement of either the TNF or Fas receptor recruited first FADD and then the proenzyme form of caspase-8. Upon treatment with an anti-Fas antibody or with TNF, hepatocytes, fibroblasts, or thymocytes derived from Bid-deficient mice activate caspase-8 but survive longer than their comparable wild-type cells (17Yin X.-M. Wang K. Gross A. Zhao Y. Zinkel S. Klocke B. Roth K.A. Korsemeyer S.J. Nature. 1999; 400: 886-891Crossref PubMed Scopus (860) Google Scholar). By contrast, other inducers of apoptosis, including staurosporine, dexamethasone, and γ-irradiation, killed Bid-deficient and wild-type cells to the same extent (17Yin X.-M. Wang K. Gross A. Zhao Y. Zinkel S. Klocke B. Roth K.A. Korsemeyer S.J. Nature. 1999; 400: 886-891Crossref PubMed Scopus (860) Google Scholar). A second model of Bax activation proposes that a change in the pH of the cytosol alters the conformation of the protein, an effect that results in exposure of the membrane-targeting C-terminal domain and translocation to the mitochondria. Withdrawal of IL-7 from T lymphocytes (D1 cells) that are dependent on this cytokine for continued viability produced a rise in intracellular pH, Bax translocation, and apoptotic cell death (15Khaled A.R. Kim K. Hofmeister R. Muegge K. Durum S.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14476-14481Crossref PubMed Scopus (217) Google Scholar). Similarly, alkalinization and Bax translocation were observed after IL-3 withdrawal from a different cell line (15Khaled A.R. Kim K. Hofmeister R. Muegge K. Durum S.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14476-14481Crossref PubMed Scopus (217) Google Scholar). More recently, U937 cells treated with C2-ceramide evidenced an early rise in intracellular pH associated with a conformational change in Bax (23Belaud-Rotureau M.A. Leducq N. Macouillard Poulletier de Gannes F. Diolez P. Lacombe F. Bernard P. Belloc F. Apoptosis. 2000; 5: 551-560Crossref PubMed Scopus (50) Google Scholar). Alkalinization itself induced Bax translocation, as demonstrated by two experiments with D1 cells maintained in IL-7 (15Khaled A.R. Kim K. Hofmeister R. Muegge K. Durum S.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14476-14481Crossref PubMed Scopus (217) Google Scholar). First, Bax translocation occurred upon incubation of homogenates in buffers of pH 7.8 or higher (15Khaled A.R. Kim K. Hofmeister R. Muegge K. Durum S.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14476-14481Crossref PubMed Scopus (217) Google Scholar). In the second experiment, nigericin was used to equilibrate intact cells with the extracellular pH. Again, Bax translocation was observed only upon incubation in alkaline buffers (15Khaled A.R. Kim K. Hofmeister R. Muegge K. Durum S.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14476-14481Crossref PubMed Scopus (217) Google Scholar). The present study considered the role of changes in intracellular pH in the mechanism of Bax activation in HeLa cells upon induction of apoptosis by either staurosporine or TNF. In both cases a change in intracellular pH was accompanied by translocation of Bax to the mitochondria with consequent release of cytochrome c and the death of the cells. The chloride channel inhibitor furosemide prevented the pH change induced by either staurosporine or TNF. At the same time, furosemide prevented Bax translocation, cytochrome crelease, and cell death. In TNF-intoxicated cells, furosemide did not prevent the cleavage of Bid. In staurosporine-intoxicated cells, furosemide did prevent the translocation of full-length Bid to the mitochondria. HeLa cells (ATCC-CC-1) were maintained in 25-cm2 polystyrene flasks with 5 ml of Dulbecco's modified Eagle's medium (DMEM; Mediatech, Inc., Hedron, VA) containing 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 10% heat-inactivated fetal bovine serum (complete DMEM). The cells were incubated under an atmosphere of 95% air and 5% CO2. For all experiments HeLa cells were plated at a density of 30,000 cells/cm2 in complete DMEM. Following overnight incubation, the cells were washed twice with PBS and placed in DMEM without serum. The day after, the cells were washed with PBS, and the medium was replaced with Hank's balanced salt solution (HBSS) without serum, which was supplemented with 2 mm l-glutamine and 1 mm glycine and adjusted to pH 7.4. Cells were incubated for 90 min at 37 °C prior to treatment. All treatments were performed in HBSS medium without serum at pH 7.4. In all experiments staurosporine (Biomol) was added to a final concentration of 150 nm. STR was dissolved in Me2SO and added to the cells in 0.2% volume. Human TNF (Calbiochem) was dissolved in sterile PBS containing 0.5% bovine serum albumin and added to the cultures at a final concentration of 10 ng/ml. Cycloheximide (Sigma) was dissolved in PBS and added at a final concentration of 10 μm. Furosemide (Sigma) was dissolved in Me2SO and added in 0.5% volume at a final concentration of 2 mm. Where indicated, the cells were pretreated for 30 min with furosemide before addition of either STR or TNF and CHX. The cell permeable caspase-8 inhibitor IETD-fluoromethyl ketone (Kamiya Biomedical Co., Seattle, WA) was dissolved in Me2SO and added in a 0.2% volume to give a final concentration of 50 μm. In all cases the vehicles used to prepare stock solutions of the reagents had no effect on the cells or the parameters measured at the concentration used. Cell viability was determined at the times indicated in the text by the release of lactate dehydrogenase into the culture medium as described previously (24Pastorino J.G. Snyder J.W. Hoek J.B. Farber J.L. Am. J. Physiol. 1995; 268: C676-C685Crossref PubMed Google Scholar). Cells (105) were plated on 12-mm glass coverslips placed in a 24-well plate. HeLa cells were treated in HBSS without serum and loaded subsequently with 1 μm(2′,7′- bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein), acetoxymethylester (BCECF-AM) (Molecular Probes, Eugene, OR) for 30 min at 37 °C. BCECF-AM was dissolved in Me2SO according to the manufacturer's instructions. Following a 30-min incubation, each coverslip was washed with PBS and transferred to a cuvette with 2 ml of HBSS. The pH-sensitive fluorescence was assessed on a PerkinElmer Life Sciences LS4 fluorescence spectrophotometer. The excitation was set alternatively at 490 or 440 nm, and the emission was set at 535 nm. The pHi values were determined from a standard curve based upon the ratio of fluorescence intensities measured at the two excitation wavelengths. HeLa cells grown on coverslips were transferred to a high K+ buffer (30 mm NaCl, 120 mm KCl, 1 mm CaCl2, 0.5 mm MgSO4, 1 mmNaH2PO4, 5 mm glucose, 10 mm HEPES, 10 mm PIPES), adjusted to various pH values between 6.0 to 8.0 in the presence of 1 μmBCECF-AM. To adjust the intracellular pH to that of the extracellular buffer, the cells were treated for 30 min at 37 °C with 10 μm nigericin (Sigma). By plotting the fluorescence ratioversus the pH values, a pH calibration curve was generated. Cells (4 × 106) were plated in 75-cm2polystyrene flasks. Following treatment the cells were harvested by centrifugation at 750 × g for 10 min at 4 °C. The cell pellets were resuspended in 1 ml of 20 mm HEPES-KOH, pH 7.5, 10 mm KCl, 1.5 mm MgCl2, 1 mm EDTA, 1 mm EGTA, 1 mmphenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 250 mm sucrose. The cells were broken open with six passages through a 26-gauge needle applied to a 1-ml syringe. The homogenate was centrifuged at 750 × g for 10 min at 4 °C to remove nuclei and unbroken cells. The supernatant was transferred to a high speed centrifuge tube. Centrifugation was conducted at 10,000 × g for 15 min at 4 °C. The resulting mitochondrial pellet was lysed in 50 μl of 20 mm Tris, pH 7.4, 100 mm NaCl, 1 mmphenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1% Triton X-100. The supernatant from the 10,000 × g spin was centrifuged at 100,000 × g(60 min at 4 °C), and the resulting supernatant was used for preparation of cytosol. The cytosolic fraction was concentrated through a Microcon YM-10 centrifugal filter device (Millipore, Bedford, MA). Protein content of each fraction was determined by the bicinchoninic acid assay (Sigma). Equivalent amounts of protein were electrophoresed on SDS-polyacrylamide gels. Kaleidoscope prestained standards (Bio-Rad) were used to determine molecular weight. The gels were then electroblotted onto nitrocellulose membranes. Bid was detected with a rabbit polyclonal antibody (BIOSOURCE, Camarillo, CA). Bax was detected with a rabbit polyclonal antibody (N-20) (Santa Cruz Biotechnology, Santa Cruz, CA). Cytochrome c was detected by a monoclonal antibody (Pharmingen). To monitor the purity of the subcellular preparations, the same nitrocellulose blots used for the determination of Bax, Bid, and cytochrome c were also stained for either COX4 (cytochrome c oxidase subunitIV) or β-actin (antihuman mouse monoclonal antibodies against either COX4 (Clontech, Palo Alto, CA) or β-actin (Santa Cruz Biotechnology), respectively). In each case, the relevant protein was visualized by staining with the appropriate secondary horseradish peroxidase-labeled antibody followed by enhanced chemiluminescence. The fluorescence excitation profile of the indicator dye BCECF is pH-dependent over the range of possible cytoplasmic pH and was, accordingly, used here for determining pHi. Treatment of HeLa cells with 150 nm staurosporine produced a rise in pHi. Fig 1 shows that an increased pHi was detected within 3 h and reached a peak of pH 8.0 after 4 h, a rise of at least 0.6 units over the initial pH. The intracellular pH remained elevated through the fifth hour after treatment with staurosporine. The chloride channel inhibitor furosemide (25Haas M. Am. J. Physiol. 1994; 267: C869-C885Crossref PubMed Google Scholar) prevented this intracellular alkalinization produced by staurosporine. Fig. 1 shows that throughout the entire 5-h duration of the experiment, pHi remained at the initial level in the presence of both staurosporine and furosemide. The proapoptotic proteins Bax and Bid are expressed constitutively in HeLa cells. Staurosporine produced a redistribution of both Bax and Bid from the cytosol to the mitochondria (6Desagher S. Osen-Sand A. Nichols A. Eskes R. Montessuit S. Lauper S. Maundrell K. Antonsson B. Martinou J.-C. J. Cell Biol. 1999; 144: 891-901Crossref PubMed Scopus (1089) Google Scholar) (Fig. 2). Fig. 2 compares the content of Bax in the mitochondria and in the cytosol within 6 h of the treatment of HeLa cells with either staurosporine alone or staurosporine and furosemide. With staurosporine alone, the content of Bax increased in the mitochondria and decreased in the cytosol. Furosemide prevented this translocation of Bax. When the HeLa cells were treated with both furosemide and staurosporine, the content of Bax in the mitochondria did not increase, and that in the cytosol decreased only slightly (Fig. 2). A similar effect of furosemide occurred with the translocation of Bid. Full-length Bid (p22 in Fig. 2) accumulated in the mitochondria in response to staurosporine. At the same time, the content of full-length Bid in the cytosol decreased (Fig. 2). The cleavage product of Bid (p15 in Fig. 2) was present in the cytosol but not detectable in the mitochondria 6 h after treatment with staurosporine alone. Furosemide prevented the staurosporine-induced increase in full-length Bid in the mitochondria and the decrease in the cytosol (Fig. 2). The accumulation of tBid in the cytosol within 6 h of treatment with staurosporine alone did not occur in the presence of furosemide (Fig.2). Importantly, the changes in the intracellular distribution of Bax and Bid cannot be interpreted as reflecting differences in either the number of mitochondria or the relative content of cytosolic proteins compared under the various conditions illustrated in Fig. 2. The content of the mitochondrial marker protein COX4 did not vary under the conditions studied (Fig. 2). Similarly, the content of the cytosolic marker protein β-actin did not vary under the conditions studied (Fig. 2). In HeLa cells treated with staurosporine, the translocation of Bax and Bid to the mitochondria is accompanied by induction of the mitochondrial permeability transition and the release of cytochrome c into the cytosol (11Tafani M. Minchenko D.A. Serroni A. Farber J.L. Cancer Res. 2001; 61: 2459-2466PubMed Google Scholar). In parallel with the inhibition of Bax and Bid translocation, furosemide reduced the loss of cytochrome c from the mitochondria and, in turn, its accumulation in the cytosol substantially (Fig.3). The content of cytochromec in the mitochondria decreased, and the content in the cytosol increased within 6 h of treatment with staurosporine (Fig.3). Furosemide reduced this change in the distribution of cytochromec (Fig. 3). Again, the purity of the mitochondrial and the cytosolic fractions was assessed by the presence of the COX4 and the β-actin proteins, respectively (Fig. 3). The biochemical effects of furosemide on staurosporine-intoxicated HeLa cells were reflected in a prevention of the cell killing. Treatment of HeLa cells with 150 nmstaurosporine killed almost 50% of the cells within 20 h (Fig.4). Addition of furosemide to the culture medium 30 min prior to treatment with staurosporine prevented this cell killing. After 20 h the number of dead cells in the presence of both furosemide and staurosporine was not significantly different from that in the control, untreated cultures (Fig. 4). The next experiments considered the effect of furosemide on the another model of apoptosis in HeLa cells. Like staurosporine, TNF has been used widely to induce apoptosis in a variety of cell types. Most cells are not killed by exposure to TNF alone. Upon inhibition of RNA or protein synthesis, however, these same cells become sensitive to this cytokine. In the presence of the protein synthesis inhibitor cycloheximide, TNF killed almost 60% of the HeLa cells within 16 h (Fig.5). Furosemide reduced the extent of this cell killing substantially. When furosemide was added to the culture medium 30 min prior to that of TNF and cycloheximide, less than 20% of the cells were dead 16 h later (Fig. 5). The fluorescent indicator dye BCECF was again used to determine the effect of TNF on the intracellular pH of HeLa cells. TableI shows that TNF in the presence of cycloheximide produced an intracellular acidification. Within 3 h of exposure to TNF, the pHi decreased by one-half pH unit to 6.6 from the initial pH observed in the control cells. This intracellular acidification was prevented by furosemide. In the presence of TNF and furosemide, pHi was essentially the same as in the controls (Table I).Table IIntracellular acidification upon treatment with TNF and its prevention by furosemideTreatmentpHiControl7.1 ± 0.2TNF + CHX6.6 ± 0.2TNF + CHX + furosemide7.2 ± 0.2HeLa cells were treated for 3 h with TNF plus cycloheximide or TNF plus cycloheximide and furosemide. Control cells were untreated. The intracellular pH (pHi) was then determined with the use of BCECF-AM as described under “Experimental Procedures.” Results are the mean ± S.D. of three separate experiments. Open table in a new tab HeLa cells were treated for 3 h with TNF plus cycloheximide or TNF plus cycloheximide and furosemide. Control cells were untreated. The intracellular pH (pHi) was then determined with the use of BCECF-AM as described under “Experimental Procedures.” Results are the mean ± S.D. of three separate experiments. As occurred with staurosporine (Fig. 2), TNF produced a translocation of Bax from the cytosol to the mitochondria. Fig.6 shows that the content of Bax in the mitochondria was increased within 4 h of the exposure of HeLa cells to TNF and cycloheximide. At the same time, the content of Bax in the cytosol decreased (Fig. 6). Furosemide reduced this translocation of Bax. When HeLa cells were pretreated with furosemide, the increase of Bax in the mitochondria was less than with TNF and cycloheximide alone (Fig. 6). Similarly, the decrease of Bax in the cytosol was less in the presence of furosemide than with TNF and cycloheximide alone (Fig. 6). Fig. 6 also shows that the content of the mitochondrial marker COX4 and the cytosolic marker β-actin did not vary under the conditions studied. A trivial explanation for the effect of furosemide on Bax translocation might be an interaction of the drug directly with Bax to change its conformation independently of a change in pHi. To rule out this possibility, control and TNF-treated cells were homogenized and fractionated in the presence or absence of furosemide. Western blots were obtained with an antibody that detected an altered Bax conformation. The presence of furosemide did not affect the content of Bax in any fraction (data not shown). In HeLa cells, the metabolism of Bax is similar in the two models of apoptosis studied here (see Figs. 2 and 6). By contrast, the metabolism of Bid is different in the two models, as is the effect of furosemide on these changes. Within 2 h of the treatment of HeLa cells with TNF and cycloheximide, full-length Bid is decreased in both the mitochondrial and cytosolic fractions (Fig.7 A). Within 4 h full-length Bid is almost undetectable in the mitochondria and depleted substantially in the cytosol (Fig. 7 B). After 2 h, this loss of full-length Bid is accompanied by the presence of its cleavage product, tBid (p15), in the mitochondria and less so in the cytosol (Fig. 7 A). After 4 h, the content of tBid in the mitochondria is less than at 2 h, and it is undetectable in the cytosol (Fig. 7 B). After 4 h the presence of furosemide had no effect on the changes in Bid metabolism produced by TNF and cycloheximide. As with TNF and cycloheximide alone, full-length Bid was essentially undetectable in the mitochondria in the presence of furosemide (Fig. 7 B). tBid could be observed in the mitochondria with furosemide, a result similar to that with TNF alone (Fig. 7 B). In the cytosol furosemide did not prevent the substantial depletion of full-length Bid, and there was similarly no detectable presence of tBid (Fig. 7 B). The depletion of Bid that occurs with TNF is slowed by furosemide. Although by 4 h, furosemide had no effect on Bid metabolism, there were differences after 2 h. In the presence of furosemide, the depletion of full-length Bid from both the mitochondria and cytosol was somewhat less than with TNF alone (Fig. 7 A). At the same time, the accumulation of tBid in the mitochondria was less than with TNF alone at 2 h (Fig. 7 A). All of the changes in Bid metabolism illustrated in Fig. 7, A and Boccurred in mitochondrial and cytosolic fractions that did not differ in their content of the mitochondrial marker COX4 or the cytosolic marker β-actin, respectively. In parallel with the inhibition of Bax translocation occurring with TNF, furosemide reduced the loss of cytochrome c from the mitochondria and, in turn, its accumulation in the cytosol (Fig. 8). The content of cytochrome c in the mitochondria decreased, and the content in the cytosol increased within 4 h of treatment with TNF and cyclohex" @default.
• W1963564407 created "2016-06-24" @default.
• W1963564407 creator A5020213704 @default.
• W1963564407 creator A5055882272 @default.
• W1963564407 creator A5064406981 @default.
• W1963564407 creator A5086445525 @default.
• W1963564407 creator A5087081177 @default.
• W1963564407 creator A5058075271 @default.
• W1963564407 date "2002-12-01" @default.
• W1963564407 modified "2023-10-14" @default.
• W1963564407 title "Regulation of Intracellular pH Mediates Bax Activation in HeLa Cells Treated with Staurosporine or Tumor Necrosis Factor-α" @default.
• W1963564407 cites W1593219141 @default.
• W1963564407 cites W1966397512 @default.
• W1963564407 cites W1985628988 @default.
• W1963564407 cites W1993206112 @default.
• W1963564407 cites W1998248849 @default.
• W1963564407 cites W2000093730 @default.
• W1963564407 cites W2003269554 @default.
• W1963564407 cites W2006805561 @default.
• W1963564407 cites W2012173926 @default.
• W1963564407 cites W2014586657 @default.
• W1963564407 cites W2020366336 @default.
• W1963564407 cites W2021678802 @default.
• W1963564407 cites W2022497572 @default.
• W1963564407 cites W2028111124 @default.
• W1963564407 cites W2031710396 @default.
• W1963564407 cites W2033873142 @default.
• W1963564407 cites W2042760321 @default.
• W1963564407 cites W2050293532 @default.
• W1963564407 cites W2050477129 @default.
• W1963564407 cites W2053509579 @default.
• W1963564407 cites W2062853648 @default.
• W1963564407 cites W2065413461 @default.
• W1963564407 cites W2073028327 @default.
• W1963564407 cites W2075477970 @default.
• W1963564407 cites W2085664253 @default.
• W1963564407 cites W2086365629 @default.
• W1963564407 cites W2091483784 @default.
• W1963564407 cites W2114001234 @default.
• W1963564407 cites W2115375806 @default.
• W1963564407 cites W2134872883 @default.
• W1963564407 cites W2136269288 @default.
• W1963564407 cites W2139439596 @default.
• W1963564407 cites W2140967934 @default.
• W1963564407 cites W2154757961 @default.
• W1963564407 cites W2158398965 @default.
• W1963564407 cites W2160664184 @default.
• W1963564407 cites W2162560719 @default.
• W1963564407 cites W2168726708 @default.
• W1963564407 cites W2188196872 @default.
• W1963564407 cites W2232615064 @default.
• W1963564407 cites W2263637300 @default.
• W1963564407 cites W2317926428 @default.
• W1963564407 cites W2329004735 @default.
• W1963564407 cites W4230656408 @default.
• W1963564407 cites W89852714 @default.
• W1963564407 doi "https://doi.org/10.1074/jbc.m208915200" @default.
• W1963564407 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12393866" @default.
• W1963564407 hasPublicationYear "2002" @default.
• W1963564407 type Work @default.
• W1963564407 sameAs 1963564407 @default.
• W1963564407 citedByCount "121" @default.
• W1963564407 countsByYear W19635644072012 @default.
• W1963564407 countsByYear W19635644072013 @default.
• W1963564407 countsByYear W19635644072014 @default.
• W1963564407 countsByYear W19635644072015 @default.
• W1963564407 countsByYear W19635644072016 @default.
• W1963564407 countsByYear W19635644072017 @default.
• W1963564407 countsByYear W19635644072018 @default.
• W1963564407 countsByYear W19635644072019 @default.
• W1963564407 countsByYear W19635644072021 @default.
• W1963564407 countsByYear W19635644072022 @default.
• W1963564407 countsByYear W19635644072023 @default.
• W1963564407 crossrefType "journal-article" @default.
• W1963564407 hasAuthorship W1963564407A5020213704 @default.
• W1963564407 hasAuthorship W1963564407A5055882272 @default.
• W1963564407 hasAuthorship W1963564407A5058075271 @default.
• W1963564407 hasAuthorship W1963564407A5064406981 @default.
• W1963564407 hasAuthorship W1963564407A5086445525 @default.
• W1963564407 hasAuthorship W1963564407A5087081177 @default.
• W1963564407 hasBestOaLocation W19635644071 @default.
• W1963564407 hasConcept C12554922 @default.
• W1963564407 hasConcept C134897140 @default.
• W1963564407 hasConcept C1491633281 @default.
• W1963564407 hasConcept C17991360 @default.
• W1963564407 hasConcept C185592680 @default.
• W1963564407 hasConcept C190283241 @default.
• W1963564407 hasConcept C195794163 @default.
• W1963564407 hasConcept C203014093 @default.
• W1963564407 hasConcept C2777366897 @default.
• W1963564407 hasConcept C2779084600 @default.
• W1963564407 hasConcept C3020084786 @default.
• W1963564407 hasConcept C502942594 @default.
• W1963564407 hasConcept C503630168 @default.
• W1963564407 hasConcept C54355233 @default.
• W1963564407 hasConcept C55493867 @default.
• W1963564407 hasConcept C62478195 @default.
• W1963564407 hasConcept C79879829 @default.

• next