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• W2010587120 abstract "The movement of intracellular monovalent cations has previously been shown to play a critical role in events leading to the characteristics associated with apoptosis. A loss of intracellular potassium and sodium occurs during apoptotic cell shrinkage establishing an intracellular environment favorable for nuclease activity and caspase activation. We have now investigated the potential movement of monovalent ions in Jurkat cells that occur prior to cell shrinkage following the induction of apoptosis. A rapid increase in intracellular sodium occurs early after apoptotic stimuli suggesting that the normal negative plasma membrane potential may change during cell death. We report here that diverse apoptotic stimuli caused a rapid cellular depolarization of Jurkat T-cells that occurs prior to and after cell shrinkage. In addition to the early increase in intracellular Na+, 86Rb+studies reveal a rapid inhibition of K+ uptake in response to anti-Fas. These effects on Na+ and K+ ions were accounted for by the inactivation of the Na+/K+-ATPase protein and its activity. Furthermore, ouabain, a cardiac glycoside inhibitor of the Na+/K+-ATPase, potentiated anti-Fas-induced apoptosis. Finally, activation of an anti-apoptotic signal,i.e. protein kinase C, prevented both cellular depolarization in response to anti-Fas and all downstream characteristics associated with apoptosis. Thus cellular depolarization is an important early event in anti-Fas-induced apoptosis, and the inability of cells to repolarize via inhibition of the Na+/K+-ATPase is a likely regulatory component of the death process. The movement of intracellular monovalent cations has previously been shown to play a critical role in events leading to the characteristics associated with apoptosis. A loss of intracellular potassium and sodium occurs during apoptotic cell shrinkage establishing an intracellular environment favorable for nuclease activity and caspase activation. We have now investigated the potential movement of monovalent ions in Jurkat cells that occur prior to cell shrinkage following the induction of apoptosis. A rapid increase in intracellular sodium occurs early after apoptotic stimuli suggesting that the normal negative plasma membrane potential may change during cell death. We report here that diverse apoptotic stimuli caused a rapid cellular depolarization of Jurkat T-cells that occurs prior to and after cell shrinkage. In addition to the early increase in intracellular Na+, 86Rb+studies reveal a rapid inhibition of K+ uptake in response to anti-Fas. These effects on Na+ and K+ ions were accounted for by the inactivation of the Na+/K+-ATPase protein and its activity. Furthermore, ouabain, a cardiac glycoside inhibitor of the Na+/K+-ATPase, potentiated anti-Fas-induced apoptosis. Finally, activation of an anti-apoptotic signal,i.e. protein kinase C, prevented both cellular depolarization in response to anti-Fas and all downstream characteristics associated with apoptosis. Thus cellular depolarization is an important early event in anti-Fas-induced apoptosis, and the inability of cells to repolarize via inhibition of the Na+/K+-ATPase is a likely regulatory component of the death process. plasma membrane potential protein kinase C phorbol 12-myristate 13-acetate carbonyl cyanide m-chlorophenylhydrazone bis-(1,3-dibutylbarbituric acid) trimethine oxonol 3,3′-dihexyloxacarbocyanine iodide sodium-binding benzofuran isophthalate acetyloxymethylester propidium iodide phosphate-buffered saline Tris-buffered saline mitochondrial membrane potential Apoptosis is a fundamental physiological process where activation of specific biochemical and morphological events results in cellular suicide. Although programmed cell death is a normal physiologic process observed during development and cellular homeostasis, insufficient or excessive apoptosis can lead to various pathological conditions, such as Alzheimer's and Parkinson's disease, cancer, and AIDS. The loss of cell volume, chromatin condensation, and internucleosomal DNA fragmentation are all defining characteristics of this mode of cell death. Recently, a loss of intracellular monovalent ions has been shown to play a pivotal role in apoptosis (1Wang L. Xu D. Lu L. J. Biol. Chem. 1999; 274: 3678-3685Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 2Yu S.P. Yeh C.-H. Strasser M. Tian M. Choi D.W. Science. 1999; 284: 336-339Crossref PubMed Scopus (210) Google Scholar, 3Dallaporta B. Hirsch T. Susin S.A. Zamzami N. Larochette N. Brenner C. Marzo I. Kroemer G. J. Immunol. 1998; 160: 5605-5615PubMed Google Scholar, 4Bilney A.J. Murray A.W. FEBS Lett. 1998; 424: 221-224Crossref PubMed Scopus (29) Google Scholar, 5Yu S.P. Farhangrazi Z.S. Ying H.S. Yeh C.-H. Choi D.W. Neurobiol. Dis. 1998; 5: 81-88Crossref PubMed Scopus (181) Google Scholar, 6Bortner C.D. Hughes Jr., F.M. Cidlowski J.A. J. Biol. Chem. 1997; 272: 32436-32442Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar, 7Hughes Jr., F.M. Bortner C.D. Purdy G.D. Cidlowski J.A. J. Biol. Chem. 1997; 272: 30567-30576Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 8Yu S.P. Yeh C.-H. Sensi S.L. Gwag B.J. Canzoniero L.M.T. Farhangrazi Z.S. Ying H.S. Tian M. Dugan L.L. Choi D.W. Science. 1997; 278: 114-117Crossref PubMed Scopus (537) Google Scholar, 9McCarthy J.V. Cotter T.G. Cell Death Differ. 1997; 4: 756-770Crossref PubMed Scopus (106) Google Scholar, 10Barbiero G. Duranti F. Bonelli G. Amenta J.S. Baccino F.M. Exp. Cell Res. 1995; 217: 410-418Crossref PubMed Scopus (141) Google Scholar, 11Jonas D. Walev I. Berger T. Liebetrau M. Palmer M. Bhakdi S. Infect. Immun. 1994; 62: 1304-1312Crossref PubMed Google Scholar). A major loss of both intracellular potassium and sodium occurs when apoptotic cells shrink and prior to the loss of membrane integrity (6Bortner C.D. Hughes Jr., F.M. Cidlowski J.A. J. Biol. Chem. 1997; 272: 32436-32442Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar, 7Hughes Jr., F.M. Bortner C.D. Purdy G.D. Cidlowski J.A. J. Biol. Chem. 1997; 272: 30567-30576Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar). Maintenance of the normal physiologic intracellular concentration of these monovalent ions was also shown to inhibit the activation of effector caspases (caspase-3-like enzymes) and the apoptotic nuclease activity during cell death, suggesting that the role ions play during apoptosis is more extensive than simply facilitating the loss of cell volume (7Hughes Jr., F.M. Bortner C.D. Purdy G.D. Cidlowski J.A. J. Biol. Chem. 1997; 272: 30567-30576Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar). In most excitable cells, cellular depolarization occurs as a result of a movement of sodium ions, which can occur through a variety of mechanisms including opening of voltage-gated sodium channels (12Kallen R.G. Cohen S.A. Barchi R.L. Mol. Neurobiol. 1993; 7: 383-428Crossref PubMed Scopus (73) Google Scholar), suppression of the Na+/K+-ATPase activity (13Jones G.S. Van Dyke K. Castranova V. J. Cell. Physiol. 1981; 106: 75-83Crossref PubMed Scopus (40) Google Scholar,14Geck P. Pietrzyk C. Burckhardt B.C. Pfeiffer B. Heinz E. Biochim. Biophys. Acta. 1980; 600: 432-447Crossref PubMed Scopus (337) Google Scholar), and activation of Na+-dependent amino acid co-transport systems, which can act like sodium ionophores (15Hacking C. Eddy A. Biochem. J. 1981; 194: 415-426Crossref PubMed Scopus (16) Google Scholar, 16Philo R.D. Eddy A. Biochem. J. 1978; 174: 801-810Crossref PubMed Scopus (47) Google Scholar). In contrast to excitable cells, lymphocytes have a relatively stable sodium concentration, and very little detail is known about the movement of sodium ions in these cells, although several studies have suggested that changes in sodium levels in lymphocytes may occur by similar mechanisms as in other cell types (17Senn N. Garay R.P. Am. J. Physiol. 1989; 257: C12-C18Crossref PubMed Google Scholar, 18Adebodun F. Post J.F. M J. Cell. Physiol. 1993; 154: 199-206Crossref PubMed Scopus (26) Google Scholar). Nonetheless, the movement of ions, especially sodium, in lymphocytes would be likely to be reflected in a change in plasma membrane potential (PMP).1 The loss of the mitochondrial membrane potential has been shown to occur in a variety of apoptotic model systems (19Inai Y. Yabuki M. Kanno T. Akiyama J. Yasuda T. Utsumi K. Cell Struct. Funct. 1997; 22: 555-563Crossref PubMed Scopus (78) Google Scholar, 20Zamzami N. Marchette P. Castedo M. Hirsch T. Susin S.A. Masse B. Kroemer G. FEBS Lett. 1996; 384: 53-57Crossref PubMed Scopus (388) Google Scholar, 21Cossarizza A. Kalashnikova G. Grassilli E. Chiappelli F. Salviolo S. Capri M. Barbieri D. Troiano L. Monti D. Franceschi C. Exp. Cell Res. 1994; 214: 323-330Crossref PubMed Scopus (186) Google Scholar). Mitochondrial depolarization is proposed to occur through the opening of permeability transition pores, located on the inner mitochondrial membrane, thus disrupting the member potential by permitting the redistribution of ions across the membrane (22Lemasters J.J. Nieminen A.-L. Qian T. Trost L.C. Elmore S.P. Nishimura Y. Crowe R.A. Cascio W.E. Bradham C.A. Brenner D.A. Herman B. Biochim. Biophys. Acta. 1998; 1366: 177-196Crossref PubMed Scopus (1227) Google Scholar, 23Bernardi P. Biochim. Biophys. Acta. 1996; 1275: 5-9Crossref PubMed Scopus (378) Google Scholar, 24Zamzami N. Marchetti P. Castedo M. Zanin C. Vayssière J.L. Petit P.X. Kroemer G. J. Exp. Med. 1995; 181: 1661-1672Crossref PubMed Scopus (1093) Google Scholar, 25Kroemer G. Petit P.X. Zamzami N. Vayssière J.-L. Mignotte B. FASEB J. 1995; 9: 1277-1287Crossref PubMed Scopus (967) Google Scholar, 26Zoratti M. Szabo I. Biochim. Biophys. Acta. 1995; 1241: 139-176Crossref PubMed Scopus (2194) Google Scholar). Recent evidence has shown that changes in the mitochondrial membrane potential, along with several other characteristics of apoptosis, appear to be restricted to the shrunken population of cells (27Bortner C.D. Cidlowski J.A. J. Biol. Chem. 1999; 274 (21952): 21953Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). These observations led us to investigate whether an early transit of monovalent ions, prior to the loss of cell volume, might promote the activation of apoptosis and lead to the downstream movement of ions and apoptotic events associated with cell death. Flow cytometry allows multiple cell death characteristics to be analyzed at the single cell level and thus has been an invaluable tool in the study of apoptosis. We have used this technology to ascertain if an early movement of monovalent ions occurs during apoptosis. By using a fluorescent dye that measures changes in intracellular sodium, we show that an early increase in intracellular sodium occurs prior to the loss of cell volume. We hypothesized that this increase in intracellular sodium would be reflected in changes in the plasma membrane potential. By using flow cytometry and a dye that responds to acute changes in the plasma membrane potential (PMP), we show that cells depolarize very early during apoptosis, prior to a loss in cell volume, and in response to various apoptotic stimuli. We also observed that changes in PMP correlated with a population of cells with increased intracellular sodium. K+ uptake studies using86Rb+ suggested the rapid inactivation of the Na+/K+-ATPase, which was confirmed by using a functional enzyme activity assay and Western blot analysis. We also show that inhibition of the Na+/K+-ATPase by using ouabain enhances cellular depolarization and apoptosis, whereas treatment with an anti-apoptotic PKC activator prevents anti-Fas-induced cellular depolarization and cell death. These studies indicate that an early movement of monovalent ions, particularly sodium, results in plasma membrane depolarization that may orchestrate subsequent movement of ions during apoptosis. Jurkat cells, E6.1 (human lymphoma), were cultured in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum, 4 mm glutamine, 31 mg/liter penicillin, and 50 mg/liter streptomycin at 37 °C, 7% CO2 atmosphere. Induction of apoptosis in Jurkat cells (5 × 105 cells per ml) was accomplished using either 10 or 50 ng/ml anti-human Fas IgM (Kamiya Biomedical), 2 μmA23187 (Calbiochem), or 10 μmthapsigargin (Sigma). The cells were incubated at 37 °C, 7% CO2 atmosphere for the specified periods. The caspase-8 inhibitor benzyloxycarbonyl-IETD-fluormethyl ketone was purchased from Kamiya Biomedical. Ouabain and the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) were purchased from Sigma. Phorbol 12-myristate 13-acetate (PMA; synthetic analog of diacylglycerol) was purchased from Calbiochem. Cell size and changes in the light scattering properties of the cell were determined by flow cytometry as described previously using a Becton Dickinson FACSort (28Bortner C.D. Cidlowski J.A. Am. J. Physiol. 1996; 271: C950-C961Crossref PubMed Google Scholar). Briefly, 7,500 cells were examined by exciting the cells with a 488 nm argon laser and determining their position on a forward-scatter versus side-scatter dot plot. Light scattered in the forward direction is roughly proportional to cell size, whereas light scattered at a 90° angle (side scatter) is proportional to cell density or granularity (29Willman C.L. Stewart C.C. Semin. Diagnostic Pathol. 1989; 6: 3-12PubMed Google Scholar). Therefore, as a cell shrinks or loses cell volume, a decrease in the amount of forward-scattered light is observed, along with a slight change in side-scattered light. A gate based on the properties of the control cells was set on each forward-scatter versus side-scatter dot plot to separate the normal and apoptotic populations of cells and remained constant throughout the analysis. Acute changes in the plasma membrane potential were measured by flow cytometry using DiBAC4(3) (Molecular Probes). DiBAC4(3) was prepared in Me2SO according to the manufacturer's instructions. Graded potassium media were made by altering the KCl and NaCl concentrations in RPMI 1640 media containing glutamine and antibiotics. The normal KCl and NaCl concentrations in RPMI 1640 are 5.4 and 102.7 mm, respectively, totaling 108.1 mm for these salts. For graded potassium media, the KCl concentration was set at 5.4 (normal), 25, 50, 75, or 102.7 mm, whereas the NaCl concentration was adjusted such that the combined monovalent salt concentration equaled 108.1 mm. Heat-inactivated fetal calf serum, dialyzed against several changes of the KCl/NaCl-free RPMI 1640, was added to a final concentration of 10%. Jurkat cells were resuspended in 1 ml of the various graded potassium media or in normal RPMI 1640 containing 150 nm DiBAC4(3) at a density of 5 × 105 cells per ml. All samples were incubated for 10 min at 37 °C, 7% CO2 atmosphere and were immediately examined by flow cytometry using a Becton Dickinson FACSort, with excitation performed using a 488 nm argon laser, and fluorescent emission was detected at 530 nm (FL-1). Ten thousand cells were examined under each condition, and all flow cytometric analyses were accomplished using CellQuest software. Jurkat cells treated with either 10 ng/ml of an anti-Fas antibody, 2 μmA23187, or 10 mmthapsigargin were incubated at 37 °C, 7% CO2atmosphere. Stock solutions of 20 μmDiBAC4(3) and DiOC6(3) (Molecular Probes) was prepared in Me2SO. Thirty minutes prior to each time of examination, either DiBAC4(3) or DiOC6(3) was added to 1 ml of cells at a final concentration of 150 ng/ml, and incubation was continued at 37 °C, 7% CO2 atmosphere. Cells were examined as changes in their plasma membrane potential by flow cytometry using either a Becton Dickinson FACSort or FACSVantage SE as described above. Ten thousand cells were examined under each condition, and all flow cytometric analyses were accomplished using CellQuest software. Jurkat cells treated with either 10 ng/ml of an anti-Fas antibody, 2 μmA23187, or 10 mm thapsigargin were incubated at 37 °C, 7% CO2 atmosphere. One hour prior to each time of examination, SBFI-AM (Na+) was added to 1 ml of cells at a final concentration of 5 μm, and incubation was continued at 37 °C, 7% CO2 atmosphere. Immediately prior to flow cytometric examination, propidium iodide (PI, Sigma) was added to a final concentration of 10 μg/ml. Ten thousand cells were analyzed by sequential excitation of the cells containing SBFI-AM and PI at 340–350 and 488 nm, respectively, using a FACSVantage SE flow cytometer (Becton Dickinson) and CellQuest software. For the86Rb+ efflux experiments, Jurkat cells (5 × 105 cells per ml) loaded overnight with 12.5 μCi of86Rb+ were washed twice in normal RPMI 1640 and then split into 2 samples at a final cell density of 1 × 106 cells per ml. Anti-Fas antibody was added to one sample at a final concentration of 100 ng/ml, and all samples were incubated at 37 °C, 7% CO2 atmosphere. At 1-h intervals, 3 separate 1-ml aliquots of cells were harvested for each sample, and 800 μl of the supernatant was removed to be counted. The pellet was washed in RPMI 1640 and finally resuspended in RPMI 1640 containing 0.5% Triton X-100. Both the pellet and supernatant were counted in triplicate, and the average 86Rb+ in the pellet fraction from two independent experiments is shown ± S.E. For the86Rb+ uptake experiments, 5 μCi of86Rb+ was added to Jurkat cells (5 × 105 cells per ml) in the presence or absence of 100 ng/ml of an anti-Fas antibody. All samples were incubated at 37 °C, 7% CO2 atmosphere. At 1-h intervals, 3 separate 1-ml aliquots of cells were harvested for each sample. The pellets were washed twice in RPMI 1640 and then resuspended in RPMI 1640 containing 0.5% Triton X-100 and counted in triplicate, and the average86Rb+ in the pellet fraction from two independent experiments is shown ± S.E. The DNA content for each sample was determined as described previously by flow cytometry (28Bortner C.D. Cidlowski J.A. Am. J. Physiol. 1996; 271: C950-C961Crossref PubMed Google Scholar). Briefly, 5 ml of cells were pelleted from the culture medium and fixed by the slow addition of cold 70% ethanol to a volume of ∼1.5 ml. The volume of each sample was adjusted to 5 ml with cold 70% ethanol, and the cells were stored at 4 °C overnight. For flow analysis, the fixed cells were pelleted, washed once in 1× phosphate-buffered saline (PBS), and stained in 1 ml of 20 μg/ml PI, 1 mg/ml RNase in 1× PBS for 20 min. Seven thousand five hundred cells were examined by flow cytometry using a Becton Dickinson FACSort by gating on an area versus width dot plot to exclude cell debris and cell aggregates. The percentage of degraded DNA was determined by the number of cells with subdiploid DNA divided by the total number of cells examined under each experimental condition. The functional expression of the Na+/K+-ATPase was assessed by the ouabain-sensitive uptake of Rb+. Cells cultured in RPMI 1640 were treated in the presence or absence of 100 ng/ml of an anti-Fas antibody for 3 h. Thirty minutes prior to the assay, 100 μm ouabain was added to the medium in the fraction of cells used for ouabain-insensitive transport. Cells were pelleted, washed with 1× PBS, and then resuspended in RPMI 1640 without K+ but containing 2.5 mm RbCl. After 10 min at 37 °C (time of transport), triplicates of 250-μl cell aliquots were immediately transferred to Eppendorf tubes on ice, centrifuged at 1000 × g for 2 min, and washed 3 times with cold 0.1m MgCl2. The pellet was resuspended in 250 μl of 0.1 m trichloroacetic acid. Cell extracts were analyzed for Rb+ by emission flame photometry in an atomic absorption spectrophotometer AA100 (PerkinElmer Life Sciences). Transport activity is expressed as μmol of Rb+ uptake per million cells in 10 min. Jurkat cells were treated in the presence or absence of 50 ng/ml of an anti-Fas antibody, harvested at the indicated times, and washed once in cold PBS. Protein extracts for each sample were prepared by resuspending the cells in a chilled lysis buffer (20 mm Tris-HCl, pH 7.5, 2 mm EDTA, 150 mm NaCl, and 0.5% Triton X-100) containing a mixture of protease inhibitors (1 μm pepstatin, 1 μmleupeptin, 1 μg/ml aprotinin, 1 μm pepstatin, and 1 mm phenylmethylsulfonyl fluoride) and were homogenized with a Dounce homogenizer. After 15 min of centrifugation at 13,000 rpm in a microcentrifuge, the supernatant was collected and assayed for protein concentration by the method of Bradford using the Bio-Rad system. 20–50 μg of protein per sample equally diluted in Laemmli loading buffer and denatured for 5 min were examined by gel electrophoresis at 120 V for 2 h using 12% SDS-polyacrylamide gel electrophoresis gels (NOVEX, San Diego, CA). The gels were then electrophoretically transferred to nitrocellulose membranes (Schleicher & Schuell) at 42 V for 1.5 h and stained with Ponceau S (Sigma) to verify the equal amount and quality of protein between lanes prior to Western blotting. Membranes were blocked overnight at 4 °C in Tris-buffered saline (TBS) containing 0.05% Tween (Sigma) and 5% nonfat dried milk. Monoclonal anti-Na+/K+recognizing human α and β isoforms (Affinity Bioreagents, Golden, CO) and monoclonal anti-caspase-8 (Calbiochem) were diluted 1:250 in TBS, 0.05% Tween, 0.5% milk and membranes were blotted with the correspondent antibody for 1 h at room temperature. Blots were washed 3 times with TBS, 0.05% Tween and incubated for 1 h with peroxidase-linked anti-mouse IgG (Amersham Pharmacia Biotech) diluted 1:5000 in TBS, 0.05% Tween, 0.5% milk. Following washes with TBS, membranes were treated with ECL chemiluminescence detection system, exposed to hyperfilm (ECL, PerkinElmer Life Sciences), and developed. The maintenance of a homeostatic balance of intracellular and extracellular ions is crucial for cell survival. Alterations in this ionic balance can signal a cell to divide, differentiate, or even to undergo cell death. We have previously shown that a dramatic loss of intracellular ions, particularly sodium and potassium, is associated with the shrinkage of cells during apoptosis, thus altering the intracellular environment and permitting nuclease activity and effector caspase activation (6Bortner C.D. Hughes Jr., F.M. Cidlowski J.A. J. Biol. Chem. 1997; 272: 32436-32442Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar, 7Hughes Jr., F.M. Bortner C.D. Purdy G.D. Cidlowski J.A. J. Biol. Chem. 1997; 272: 30567-30576Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar). In this study, we were interested in determining if a change in monovalent cations could be detected in response to apoptotic stimulation, prior to cell shrinkage. Thus, we analyzed Jurkat cells treated with anti-Fas for changes in intracellular sodium using the sodium-binding fluorescent indicator SBFI-AM (Na+) (6Bortner C.D. Hughes Jr., F.M. Cidlowski J.A. J. Biol. Chem. 1997; 272: 32436-32442Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar). Flow cytometric analysis of Jurkat cells treated with an anti-Fas antibody showed a time-dependent increase in a population of cells that had an increase in intracellular sodium, which occurred prior to the loss of membrane integrity (Fig.1). Interestingly, we failed to detect any change in intracellular potassium in these cells (data not shown). Therefore, we hypothesized that this increase in intracellular sodium may be reflected in a change in the plasma membrane potential. We initially used the plasma membrane-specific dye, DiBAC4(3), to examine apoptotic cells for changes in their PMP at the single cell level by flow cytometry. DiBAC4(3) is an anionic oxonal dye that responds with an increase in fluorescent intensity at 530 nm upon membrane depolarization. We determined the utility of this membrane potential dye in our model system, Jurkat T-cells, by analyzing cells for acute changes in their PMP. Jurkat cell were depolarized with increasing concentrations of extracellular KCl. In the presence of DiBAC4(3), these KCl-treated Jurkat cells responded with a stepwise increase in DiBAC4(3) fluorescence, indicating cellular depolarization (Fig.2). To determine the specificity of this dye to measure changes specific to the PMP, we examined the response of DiBAC4(3) to acute changes in the mitochondrial membrane potential (MMP) by using various concentrations of the protonophore CCCP to collapse the membrane potential of these organelles. We have previously shown that the concentrations of CCCP used in this study were effective in uncoupling the MMP when either JC-1, a mitochondrial membrane specific dye, or DiOC6(3), a dye which responds to both changes in the mitochondrial and plasma membrane potential, were used to access changes in the MMP (27Bortner C.D. Cidlowski J.A. J. Biol. Chem. 1999; 274 (21952): 21953Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). In contrast to the results shown for acute plasma membrane depolarization, DiBAC4(3) did not respond to changes in the mitochondrial membrane potential indicating a distinct ability of DiBAC4(3) to measure strictly changes in the PMP (Fig. 2). To determine whether changes in the PMP occur during apoptosis, Jurkat cells were treated with an anti-Fas antibody, the calcium ionophoreA23187, or thapsigargin, all known apoptotic agents that differ in their mode of cell death activation (27Bortner C.D. Cidlowski J.A. J. Biol. Chem. 1999; 274 (21952): 21953Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). Under each apoptotic condition, a population of cells with an increase in DiBAC4(3) fluorescence, indicating plasma membrane depolarization, was observed in a time-dependent manner (Fig. 3). Interestingly, the observed cellular depolarization was not a transient event, as might occur in electrically excitable cells, but rather was sustained, as the population of cells with increased DiBAC4(3) fluorescence increased over time. This sustained cellular depolarization suggests that upon apoptotic stimulation, the ability of cells to repolarize is lost, thus maintaining a constant state of depolarization throughout the cell death process. In addition to the time-dependent nature of this cellular depolarization observed during cell death, we determined that this event was also sensitive to the concentration of apoptotic stimulus employed. Increasing concentrations of anti-Fas antibody added to Jurkat cells 3 h prior to flow cytometric examination in the presence of DiBAC4(3) resulted in a concentration-dependent increase in the number of cells with increased DiBAC4(3) fluorescence (Fig. 4 A), thus indicating that cellular depolarization is intrinsically linked to the degree of apoptotic stimulation. The onset of plasma membrane depolarization is rapid, occurring between 1 and 2 h after stimulation with anti-Fas (Fig. 4 B).Figure 4Response of DiBAC4(3) to measure changes in the plasma membrane potential upon increasing concentrations of anti-Fas antibody after 3 h. A, Jurkat cells treated with 10, 25, 50, or 100 ng/ml anti-Fas were incubated at 37 °C, 7% CO2 atmosphere for 2.5 h. At this time, DiBAC4(3) was added to 1 ml of cells to a final concentration of 150 ng/ml, and incubation was continued at 37 °C, 7% CO2 atmosphere for an additional 30 min. Flow cytometric analysis showed an increase in DiBAC4(3) fluorescence, indicating cellular depolarization occurred in a concentration-dependent manner. The bar graph shows the results of 3 independent experiments ± S.E.B, a time course of Jurkat cells treated with 50 ng/ml anti-Fas in the presence of DiBAC4(3) showed a rapid, time-dependent increase in the number of depolarized cells. The graph shows the results of 3 independent experiments ± S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Many characteristics of apoptosis such as changes in the mitochondrial membrane potential, the loss of intracellular ions, effector caspase activation, and DNA degradation have been shown to be restricted to the shrunken population of cells (6Bortner C.D. Hughes Jr., F.M. Cidlowski J.A. J. Biol. Chem. 1997; 272: 32436-32442Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar, 7Hughes Jr., F.M. Bortner C.D. Purdy G.D. Cidlowski J.A. J. Biol. Chem. 1997; 272: 30567-30576Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 27Bortner C.D. Cidlowski J.A. J. Biol. Chem. 1999; 274 (21952): 21953Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 28Bortner C.D. Cidlowski J.A. Am. J. Physiol. 1996; 271: C950-C961Crossref PubMed Google Scholar). Thus, we determined if cellular depolarization was also restricted to the shrunken apoptotic cells or if it occurred prior to the loss of cell volume. Flow cytometry, which permits the simultaneous examination of multiple cellular characteristics at the single cell level, was used to determine the relationship between cellular depolarization and cell size by examining DiBAC4(3) fluorescence and the forward light scattering property of the cell, respectively. When control Jurkat cells were examined on a forward-scatter versus side-scatter dot plot in the presence of DiBAC4(3), a single major population of cells was observed (Fig. 5). Gating on this single population of cells, we examined these cells on a forward-scatterversus a DiBAC4(3) fluorescence contour plot. Analysis of these control Jurkat cells showed only a single level of DiBAC4(3) fluorescence. In contrast, gating on the minor population of cells with a decrease in forward-scattered light in the control sample, denoting cells with a decreased cell size, showed that the shrunken cells had an increase in DiBAC4(3) fluorescence or a depolarized plasma membrane (Fig. 5)." @default.
• W2010587120 created "2016-06-24" @default.
• W2010587120 creator A5001320954 @default.
• W2010587120 creator A5001523226 @default.
• W2010587120 creator A5037426636 @default.
• W2010587120 date "2001-02-01" @default.
• W2010587120 modified "2023-10-15" @default.
• W2010587120 title "Plasma Membrane Depolarization without Repolarization Is an Early Molecular Event in Anti-Fas-induced Apoptosis" @default.
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