Produção Nacional
Revisado por pares
Effect of Bcl-2 Overexpression on Mitochondrial Structure and Function
2002; Elsevier BV; Volume: 277; Issue: 45 Linguagem: Inglês
10.1074/jbc.m207765200
ISSN1083-351X
AutoresAlicia J. Kowaltowski, Ricardo G. Cosso, Cláudia Barbosa Ladeira de Campos, Gary Fiskum,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoOverexpression of the antiapoptotic Bcl-2 protein enhances the uptake of fluorimetric dyes sensitive to mitochondrial membrane potential, suggesting that Bcl-2 changes the mitochondrial proton gradient. In this study, we performed calibrated measurements of mitochondrial respiration, membrane potential, ΔpH, and intramitochondrial [K+] in digitonin-permeabilized PC12 and GT1-7 neural cells that either do not express human Bcl-2 (control transfectants) or that were transfected with and overexpressed the human bcl-2gene to evaluate whether Bcl-2 alters mitochondrial inner membrane ion transport. We found that although Bcl-2-overexpressing cells exhibit higher fluorescence responses to membrane potential, pH, and K+-sensitive dyes, this increased response is due to an enhanced accumulation of these dyes and not an increased mitochondrial membrane potential, ΔpH, or [K+]. This result is supported by the presence of equal respiratory rates in Bcl-2+ and Bcl-2− cells. Possible structural alterations in Bcl-2+ mitochondria that could account for increases in fluorescent dye uptake were evaluated using flow cytometry particle sizing and light scattering determinations. These experiments established that Bcl-2-overexpressing mitochondria present both increased volume and structural complexity. We suggest that increased mitochondrial volume and structural complexity in Bcl-2+ cells may be related to many of the effects of this protein involved in the prevention of cell death. Overexpression of the antiapoptotic Bcl-2 protein enhances the uptake of fluorimetric dyes sensitive to mitochondrial membrane potential, suggesting that Bcl-2 changes the mitochondrial proton gradient. In this study, we performed calibrated measurements of mitochondrial respiration, membrane potential, ΔpH, and intramitochondrial [K+] in digitonin-permeabilized PC12 and GT1-7 neural cells that either do not express human Bcl-2 (control transfectants) or that were transfected with and overexpressed the human bcl-2gene to evaluate whether Bcl-2 alters mitochondrial inner membrane ion transport. We found that although Bcl-2-overexpressing cells exhibit higher fluorescence responses to membrane potential, pH, and K+-sensitive dyes, this increased response is due to an enhanced accumulation of these dyes and not an increased mitochondrial membrane potential, ΔpH, or [K+]. This result is supported by the presence of equal respiratory rates in Bcl-2+ and Bcl-2− cells. Possible structural alterations in Bcl-2+ mitochondria that could account for increases in fluorescent dye uptake were evaluated using flow cytometry particle sizing and light scattering determinations. These experiments established that Bcl-2-overexpressing mitochondria present both increased volume and structural complexity. We suggest that increased mitochondrial volume and structural complexity in Bcl-2+ cells may be related to many of the effects of this protein involved in the prevention of cell death. membrane potential acetoxymethyl ester 2′,7′-bis(2-carboxyethyl)-5(and -6)-carboxyfluorescein carbonyl cyanide m-chlorophenylhydrazone forward scattering side scattering The Bcl-2 protein, originally described in lymphoma cells (1Tsujimoto Y. Ikegaki N. Croce C.M. Oncogene. 1987; 2: 3-7PubMed Google Scholar) and then found to be widely distributed in a variety of cancerous tissues (2Reed J.C. Curr. Opin. Oncol. 1995; 7: 541-546Crossref PubMed Scopus (487) Google Scholar, 3Adams J.M. Cory S. Trends Biochem. Sci. 2001; 26: 61-66Abstract Full Text Full Text PDF PubMed Scopus (815) Google Scholar), is a potent inhibitor of cell death, both programmed and accidental (4Kane D.J. Sarafian T.A. Anton R. Hahn H. Gralla E.B. Valentine J.S. Ord T. Bredesen D.E. Science. 1993; 262: 1274-1277Crossref PubMed Scopus (1615) Google Scholar, 5Kroemer G. Dallaporta B. Resche-Rigon M. Annu. Rev. Physiol. 1998; 60: 619-642Crossref PubMed Scopus (1768) Google Scholar). Bcl-2 is located in biological membranes, including mitochondria (6Monaghan P. Robertson D. Amos T.A. Dyer M.J. Mason D.Y. Greaves M.F. J. Histochem. Cytochem. 1992; 40: 1819-1825Crossref PubMed Scopus (336) Google Scholar, 7Krajewski S. Tanaka S. Takayama S. Schibler M.J. Fenton W. Reed J.C. Cancer Res. 1993; 53: 4701-4714PubMed Google Scholar), and acts to inhibit mitochondrially controlled steps leading to cell death. The effects of Bcl-2 on mitochondrial control of cell death are variable according to the experimental conditions studied, indicating a multifunctional role for this protein. For example, Bcl-2 inhibits mitochondrial permeability transition (8Zamzami N. Susin S.A. Marchetti P. Hirsch T. Gomez-Monterrey I. Castedo M. Kroemer G. J. Exp. Med. 1996; 183: 1533-1544Crossref PubMed Scopus (1268) Google Scholar, 9Shimizu S. Eguchi Y. Kamiike W. Funahashi Y. Mignon A. Lacronique V. Matsuda H. Tsujimoto Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1455-1459Crossref PubMed Scopus (355) Google Scholar, 10Kowaltowski A.J. Vercesi A.E. Fiskum G. Cell Death Differ. 2000; 7: 903-910Crossref PubMed Scopus (140) Google Scholar), a process often associated with mitochondrial cytochrome c release and subsequent cell death (8Zamzami N. Susin S.A. Marchetti P. Hirsch T. Gomez-Monterrey I. Castedo M. Kroemer G. J. Exp. Med. 1996; 183: 1533-1544Crossref PubMed Scopus (1268) Google Scholar, 11Kowaltowski A.J. Castilho R.F. Vercesi A.E. FEBS Lett. 2001; 495: 12-15Crossref PubMed Scopus (716) Google Scholar), and Bcl-2 is also capable of inhibiting cytochrome c release pathways independent of mitochondrial permeability transition, such as Bid- and Bax-mediated cytochrome c release (12Polster B.M. Kinnally K.W. Fiskum G. J. Biol. Chem. 2001; 276: 37887-37894Abstract Full Text Full Text PDF PubMed Google Scholar, 13Jurgensmeier J.M. Xie Z. Deveraux Q. Ellerby L. Bredesen D. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4997-5002Crossref PubMed Scopus (1375) Google Scholar). Bcl-2 effects on mitochondria determined to date involve almost exclusively studies conducted under conditions leading to cell death. Although these studies are essential to understand the antiapoptotic effects of this protein, it is important to determine whether Bcl-2 can affect mitochondrial function under basal conditions. Understanding the changes promoted by Bcl-2 on mitochondrial function in healthy cells may determine how these cells respond to potentially deadly stimuli and uncover the common roots of the distinct antiapoptotic effects of this protein. Furthermore, Bcl-2 may have an unknown role in the regulation of basal mitochondrial energy metabolism. In fact, some previous data from both our group and others (9Shimizu S. Eguchi Y. Kamiike W. Funahashi Y. Mignon A. Lacronique V. Matsuda H. Tsujimoto Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1455-1459Crossref PubMed Scopus (355) Google Scholar, 10Kowaltowski A.J. Vercesi A.E. Fiskum G. Cell Death Differ. 2000; 7: 903-910Crossref PubMed Scopus (140) Google Scholar) suggest that Bcl-2 may regulate mitochondrial proton transport across the inner membrane resulting in higher mitochondrial membrane potentials since Bcl-2-overexpressing mitochondria take up larger quantities of membrane potential-sensitive dyes. This increased mitochondrial membrane potential could explain other Bcl-2 effects such as increased H2O2 generation (14Esposti M.D. Hatzinisiriou I. McLennan H. Ralph S. J. Biol. Chem. 1999; 274: 29831-29837Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 15Steinman H.M. J. Biol. Chem. 1995; 270: 3487-3490Abstract Full Text Full Text PDF PubMed Google Scholar, 16Armstrong J.S. Jones D.P. FASEB J. 2002; 16: 1263-1265Crossref PubMed Scopus (206) Google Scholar) since mitochondrial reactive oxygen species generation is strongly inhibited at lower membrane potentials (17Korshunov S.S. Skulachev V.P. Starkov A.A. FEBS Lett. 1997; 416: 15-18Crossref PubMed Scopus (1413) Google Scholar). Unfortunately, the cause of these mitochondrial changes promoted by Bcl-2 has never been carefully studied, and these results are often obtained from a single transfected cell line, raising the possibility that these are not universal Bcl-2 effects. In this study, we investigated the effects of Bcl-2 on mitochondrial function and structure using two separate cell lines and found that overexpression of this protein does not affect the membrane potential, respiration, ΔpH, or intramitochondrial [K+] but does increase mitochondrial volume and structural complexity. This increase in volume and structural complexity explains changes in fluorimetric membrane potential determinations conducted previously. Based on our results, we propose a model in which enhanced mitochondrial volume and structural complexity mediate many of the Bcl-2 effects related to the prevention of cell death. PC12 pheochromocytoma and immortalized hypothalamic GT1-7 neuronal cell lines transfected with the humanbcl-2 gene (Bcl-2+) or with a control retroviral construct (Bcl-2−) were maintained as described previously (4Kane D.J. Sarafian T.A. Anton R. Hahn H. Gralla E.B. Valentine J.S. Ord T. Bredesen D.E. Science. 1993; 262: 1274-1277Crossref PubMed Scopus (1615) Google Scholar). Prior to the experiments the cells were trypsinized and suspended in growth medium supplemented with 10 mm Hepes, pH 7.0. Suspended cells were kept at room temperature for up to 5 h. Cell viability, as assessed by a cell count in trypan blue, was above 95% even after 5 h at room temperature. The suspended cells were centrifuged and resuspended in the medium used in the experiment just prior to each determination. Cell protein content was determined using the Biuret method. All experiments were conducted at 37 °C. Mitochondrial ΔΨ1 was estimated through fluorescence changes of safranin O (5 mm) at excitation and emission wavelengths of 485 and 586 nm, respectively (10Kowaltowski A.J. Vercesi A.E. Fiskum G. Cell Death Differ. 2000; 7: 903-910Crossref PubMed Scopus (140) Google Scholar, 18Fiskum G. Kowaltowski A.J. Andreyev A.Y. Kushnareva Y.E. Starkov A.A. Methods Enzymol. 2000; 322: 222-234Crossref PubMed Google Scholar). Data were calibrated using a K+ gradient as described by Akerman and Wikstrom (19Akerman K.E. Wikstrom M.K. FEBS Lett. 1976; 68: 191-197Crossref PubMed Scopus (671) Google Scholar), and the membrane potential obtained for each K+ concentration was determined using the Nernst equation assuming intramitochondrial [K+] to be 150 mm, a value quite close to the experimentally determined [K+] in GT1-7 cells (see Fig. 3). A calibration curve was constructed and fitted using Origin® software, and all subsequent fluorescence traces were transformed into ΔΨ using the same fitting equation. Cells (10 mg/ml) were suspended in medium containing 10 μm BCECF-AM, 250 mmsucrose, 5 mm pyruvate, 5 mm malate, 5 mm glutamate, 100 μm EGTA, 1 mg/ml bovine serum albumin, 0.001% or 0.004% digitonin (GT1-7 and PC12 cells, respectively), and 10 mm Hepes, pH 7.2 (KOH) and incubated at 25 °C for 20 min. The permeabilized cells with BCECF-loaded mitochondria were then diluted to 2 mg/ml in 4 °C buffer devoid of BCECF, centrifuged, and resuspended in the same medium. BCECF fluorescence emission was measured at 550 nm with variable excitation wavelengths. Intramitochondrial pH was calculated from the ratio between fluorescence levels at 509 and 450 nm as described by Molecular Probes and Jung et al. (20Jung D.W. Apel L. Brierley G.P. Biochemistry. 1990; 29: 4121-4128Crossref PubMed Scopus (67) Google Scholar,21Jung D.W. Baysal K. Brierley G.P. J. Biol. Chem. 1995; 270: 672-678Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Cells were treated with CCCP and nigericin (1 μm), and the extracellular medium pH (measured using a standard pH meter) was manipulated between 7 and 8 by adding HCl and KOH. A plot relating the measured pH to the 509/450 nm fluorescence ratio was used to determine intramitochondrial pH in the absence of ionophores. All experiments were conducted within 30 min of mitochondrial loading with BCECF. Cells (10 mg/ml) were suspended in medium containing 20 μm PBFI-AM (a K+ indicator marketed by Molecular Probes) and treated in the same manner as those loaded with BCECF. PBFI fluorescence emission was measured at 500 nm with variable excitation wavelengths. Intramitochondrial [K+] was calculated from the ration between fluorescence levels at 320 and 360 nm as described by Molecular Probes and Jung et al. (20Jung D.W. Apel L. Brierley G.P. Biochemistry. 1990; 29: 4121-4128Crossref PubMed Scopus (67) Google Scholar, 21Jung D.W. Baysal K. Brierley G.P. J. Biol. Chem. 1995; 270: 672-678Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Cells were treated with CCCP and nigericin (1 μm), and spectra were collected in the presence of varying medium [K+] (50–200 mm). A plot relating [K+] to the fluorescence ratio was used to determine intramitochondrial [K+] in the absence of ionophores. Mitochondria were isolated from digitonin-permeabilized GT1-7 and PC12 cells exactly as described by Moreadith and Fiskum (22Moreadith R.W. Fiskum G. Anal. Biochem. 1984; 137: 360-367Crossref PubMed Scopus (171) Google Scholar) in isolation buffer containing 210 mm mannitol, 75 mm sucrose, 1 mg/ml bovine serum albumin, 5 mm Hepes, and 1 mm EGTA, pH 7.2 (KOH). Isolated mitochondria (∼0.2 mg/ml) were incubated in 250 mmsucrose, 10 mm Hepes, 100 μm EGTA, pH 7.2 (KOH), containing 1 μm rotenone and 5 mmK+ succinate. The suspension was analyzed by a Becton Dickinson FACSCalibur flow cytometer, and detected with a 488 nm laser. Particle size (forward scattering (FSC)) and light scattering (side scattering (SSC)) characteristics were analyzed using CellQuest software. BCECF-AM and PBFI-AM were purchased from Molecular Probes. Safranin O, EGTA, digitonin, malate, glutamate, pyruvate, bovine serum albumin, CCCP, nigericin, and valinomycin were from Sigma. Data presented as traces are representative of at least three similar repetitions. Averages represented in bar graphs were calculated from data collected in at least three repetitions using different cell preparations. Error bars indicate standard errors (S.E.), and significant differences were calculated using pairwise Tukey tests conducted by SigmaStat®. Previous studies have shown that Bcl-2 overexpression causes an increase in the uptake of fluorescent dyes sensitive to the mitochondrial ΔΨ, a result interpreted as an increase in ΔΨ induced by this protein (9Shimizu S. Eguchi Y. Kamiike W. Funahashi Y. Mignon A. Lacronique V. Matsuda H. Tsujimoto Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1455-1459Crossref PubMed Scopus (355) Google Scholar, 10Kowaltowski A.J. Vercesi A.E. Fiskum G. Cell Death Differ. 2000; 7: 903-910Crossref PubMed Scopus (140) Google Scholar). This ΔΨ effect could explain many changes observed in Bcl-2-overexpressing mitochondria, including increased reactive oxygen species generation (14Esposti M.D. Hatzinisiriou I. McLennan H. Ralph S. J. Biol. Chem. 1999; 274: 29831-29837Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 15Steinman H.M. J. Biol. Chem. 1995; 270: 3487-3490Abstract Full Text Full Text PDF PubMed Google Scholar, 16Armstrong J.S. Jones D.P. FASEB J. 2002; 16: 1263-1265Crossref PubMed Scopus (206) Google Scholar), enhanced Ca2+ uptake capacity, and larger quantities of reduced pyridine nucleotides (10Kowaltowski A.J. Vercesi A.E. Fiskum G. Cell Death Differ. 2000; 7: 903-910Crossref PubMed Scopus (140) Google Scholar, 14Esposti M.D. Hatzinisiriou I. McLennan H. Ralph S. J. Biol. Chem. 1999; 274: 29831-29837Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 23Ellerby L.M. Ellerby H.M. Park S.M. Holleran A.L. Murphy A.N. Fiskum G. Kane D.J. Testa M.P. Kayalar C. Bredesen D.E. J. Neurochem. 1996; 67: 1259-1267Crossref PubMed Scopus (201) Google Scholar). To understand the mechanism through which Bcl-2 apparently enhances ΔΨ, we performed measurements of mitochondrial uptake of the ΔΨ-sensitive probe safranin O and calibrated the data by using K+ gradients and applying the Nernst equation (see "Experimental Procedures" and Ref. 19Akerman K.E. Wikstrom M.K. FEBS Lett. 1976; 68: 191-197Crossref PubMed Scopus (671) Google Scholar). These experiments were conducted using cultured cells in which low digitonin concentrations were added to selectively permeabilize the plasma membrane, promoting a large dilution of cytosolic components while maintaining cell architecture and mitochondrial function unaltered (18Fiskum G. Kowaltowski A.J. Andreyev A.Y. Kushnareva Y.E. Starkov A.A. Methods Enzymol. 2000; 322: 222-234Crossref PubMed Google Scholar). This is the preferred method to study the effects of Bcl-2 in mitochondria from transfected cell lines since mitochondrial isolation may promote damage to the organelle in a Bcl-2-inhibited manner (18Fiskum G. Kowaltowski A.J. Andreyev A.Y. Kushnareva Y.E. Starkov A.A. Methods Enzymol. 2000; 322: 222-234Crossref PubMed Google Scholar). As noted previously (9Shimizu S. Eguchi Y. Kamiike W. Funahashi Y. Mignon A. Lacronique V. Matsuda H. Tsujimoto Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1455-1459Crossref PubMed Scopus (355) Google Scholar, 10Kowaltowski A.J. Vercesi A.E. Fiskum G. Cell Death Differ. 2000; 7: 903-910Crossref PubMed Scopus (140) Google Scholar), PC12 pheochromocytoma cells overexpressing human Bcl-2 (Bcl-2+ cells) decrease safranin fluorescence more intensely than control transfectant cells (Bcl-2-) and present an enhanced difference in fluorescence in the presence and absence of the proton ionophore CCCP (ΔFluorescence) when respiring on NADH-linked substrates (Fig. 1, upper andlower left), an effect compatible with a higher ΔΨ. A similar increase in safranin ΔFluorescence was observed in a second transfected cell line (GT1-7 hypothalamic tumor cells; Fig. 1,lower left). However, when we calibrated ΔΨ using a K+ distribution curve (Fig. 1, upper panels, see "Experimental Procedures" and Ref. 19Akerman K.E. Wikstrom M.K. FEBS Lett. 1976; 68: 191-197Crossref PubMed Scopus (671) Google Scholar), we found that Bcl-2+ mitochondria presented larger safranin responses to equal ΔΨ changes (more change in fluorescence with equal K+additions). By using the best fittings for the fluorescenceversus ΔΨ plots (Fig. 1, upper right), we were able to estimate Bcl-2− and Bcl-2+ ΔΨ in the absence of added K+ and found these to be equal in both cell lines studied (Fig. 1, lower left). Thus, Bcl-2 increases safranin fluorescence changes dependent on ΔΨ, but this effect seems to be related to an altered calibration curve and not enhanced ΔΨ. We also determined mitochondrial respiratory rates in Bcl-2+ and Bcl-2− cells (Fig. 1, lower right) and plotted them against the measured membrane potential in the presence of increasing K+ and CCCP concentrations. A change in the linear ΔΨ/respiration plot would be indicative of altered proton pumping/oxygen consumption ratios at the mitochondrial respiratory chain as proposed previously to explain the apparent higher ΔΨ in Bcl-2+ mitochondria (9Shimizu S. Eguchi Y. Kamiike W. Funahashi Y. Mignon A. Lacronique V. Matsuda H. Tsujimoto Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1455-1459Crossref PubMed Scopus (355) Google Scholar). We found that Bcl-2 does not change the correlation between mitochondrial oxygen consumption and H+pumping, a result which supports the finding that mitochondrial ΔΨ is equal in Bcl-2+ and Bcl-2− cells. Safranin is a lipophilic cation that accumulates within or in near proximity to the mitochondrial inner membrane, reducing the fluorescence of the suspension in a manner proportional to the negative charge of the mitochondrial matrix (19Akerman K.E. Wikstrom M.K. FEBS Lett. 1976; 68: 191-197Crossref PubMed Scopus (671) Google Scholar). Thus, safranin fluorescence traces measure only changes in charge across the inner membrane and are insensitive to a second component of the mitochondrial H+gradient, ΔpH. In addition, the estimated ΔΨ calculated using the Nernst equation in Fig. 1 assumes intramitochondrial K+concentrations to be ∼150 mm and equal in Bcl-2+ and Bcl-2− cells. To ascertain that Bcl-2 affects safranin distribution and not the mitochondrial proton gradient, we measured both ΔpH and K+ concentrations in Bcl-2+ and Bcl-2− mitochondria. The experiments shown in Fig. 2 compare ΔpH levels in Bcl-2− and Bcl-2+ mitochondria. We found that the addition of nigericin, a K+/H+ exchanger that reduces ΔpH and increases ΔΨ, promotes very similar effects on ΔΨ measured by calibrated safranin fluorescence in Bcl-2− and Bcl-2+ mitochondria (Fig. 2, upper panels). To confirm that Bcl-2 did not affect ΔpH, we loaded GT1-7 mitochondria with the esterified form of the pH-sensitive dye BCECF. PC12 mitochondria were not used in this experiment since they loaded very poorly with this dye, and the final fluorescence levels were insufficient to accurately estimate ΔpH. In GT1-7 Bcl-2+ cells, BCECF fluorescence was more intense and responded more significantly to the addition of nigericin than that in Bcl-2− cells (Fig. 2, bottom left). However, by calibrating the fluorescence traces (see "Experimental Procedures"), we found no difference in intramitochondrial pH levels in Bcl-2− and Bcl-2+ mitochondria despite a consistently higher BCECF load (Fig. 2, bottom right). These results indicated that, although GT1-7 mitochondria are more intensely loaded with BCECF, there is no difference in ΔpH between Bcl-2− and Bcl-2+ mitochondria. Intramitochondrial K+ levels were determined in Fig.3 by loading mitochondria with the K+ probe PBFI-AM. Again PC12 cells loaded very poorly with the dye, so only GT1-7 cells were used. We found that, although Bcl-2+ mitochondria loaded more dye and presented more intense fluorescence (Fig. 3, upper panels and lower right), no difference in intramitochondrial K+ concentrations could be detected when the data were calibrated (lower left). Indeed, intramitochondrial K+ concentrations determined using PBFI were quite close to the estimated K+ concentrations used to calibrate ΔΨ determinations in Figs. 1 and 2, ensuring the accuracy of our ΔΨ estimation. In the absence of any difference in ΔΨ, ΔpH, or intramitochondrial [K+], the increased fluorescence response to three different dyes observed in the Bcl-2+ mitochondria suggests the presence of a larger mitochondrial membrane surface (to increase safranin distribution since safranin accumulates in close contact to or within the inner membrane, Ref. 19Akerman K.E. Wikstrom M.K. FEBS Lett. 1976; 68: 191-197Crossref PubMed Scopus (671) Google Scholar) and matrix volume (to increase intramitochondrial BCECF and PBFI accumulation). To investigate this surprising possibility, we isolated mitochondria from GT1-7 and PC12 Bcl-2− and Bcl-2+ cells and evaluated their size and structural complexity using flow cytometry (Fig.4). FSC measurements using a flow cytometer can be used to estimate particle size since the intensity of light scattered at small angles from an incident laser beam is proportional to particle volume as demonstrated by Mullaney et al. (24Mullaney P.F. Van Dilla M.A. Coulter J.R. Dean P.N. Rev. Sci. Instrum. 1969; 40: 1029-1032Crossref PubMed Scopus (129) Google Scholar). In the top panels of Fig. 4, we compared FSC in Bcl-2− and Bcl-2+ mitochondria isolated from GT1-7 cells. We found that Bcl-2+ mitochondria present two populations of distinct sizes and that the average particle size of these mitochondria is larger than Bcl-2− mitochondria (Fig. 4, top left). In addition, flow cytometry can determine mitochondrial structural complexity as measured by particle light scattering (SSC), which is dependent on the refractive index of each particle. Side scattering measurements show that GT1-7 Bcl-2+ mitochondria present increased structural complexity in relation to Bcl-2− mitochondria (Fig. 4, top right). In PC12 cells, mitochondrial volume and complexity increases in Bcl-2+ cells were less pronounced but still evident (Fig. 4, lower panels). The increase in both mitochondrial size and complexity, as determined by increases in forward and side scattering, excludes the possibility that the difference between Bcl-2+ and Bcl-2− mitochondria is due to membrane damage promoted by mitochondrial isolation since mitochondria with more permeable membranes present increased size and decreased light scattering (25Garlid K.D. Beavis A.D. J. Biol. Chem. 1985; 260: 13434-13441Abstract Full Text PDF PubMed Google Scholar). Thus, in two distinct cell lines, Bcl-2 overexpression enhanced the mean size and complexity of mitochondria. The enhanced size and structural complexity of Bcl-2+ mitochondria explain why these organelles present larger responses to fluorescent dyes without changes in the mitochondrial function these dyes measure. In this study, we examined the effects of Bcl-2 overexpression on mitochondrial energetics and structure using two unrelatedbcl-2-transfected cell lines. We found that Bcl-2+ mitochondria, previously thought to present higher ΔΨ due to increased uptake of membrane potential probes such as safranin and rhodamine 123 (9Shimizu S. Eguchi Y. Kamiike W. Funahashi Y. Mignon A. Lacronique V. Matsuda H. Tsujimoto Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1455-1459Crossref PubMed Scopus (355) Google Scholar, 10Kowaltowski A.J. Vercesi A.E. Fiskum G. Cell Death Differ. 2000; 7: 903-910Crossref PubMed Scopus (140) Google Scholar), do not show any difference in ΔΨ when this measurement is calibrated using K+ gradients (Fig. 1). Instead Bcl-2+ mitochondria present a larger ability to promote fluorescence changes not only of the membrane potential probe safranin but also of pH- and K+-sensitive probes BCECF and PBFI (Figs. 2 and 3) without apparent changes in ΔΨ, ΔpH, or intramitochondrial [K+]. This finding indicates that studies comparing non-calibrated fluorescence responses in cell lines with different Bcl-2 expression levels may, in fact, misinterpret fluorescence signals and should be carefully reevaluated. Previous studies involving isolated mitochondria suggest that the increase in response promoted by Bcl-2 in fluorescence ΔΨ measurements is not due to a larger number of mitochondria in Bcl-2-overexpressing cells (26Murphy A.N. Bredesen D.E. Cortopassi G. Wang E. Fiskum G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9893-9898Crossref PubMed Scopus (386) Google Scholar), a result supported by the fact that no difference in respiratory activity can be measured between Bcl-2+ and Bcl-2− cells (Fig. 1, lower left). This observation suggests that Bcl-2+ mitochondria present both a more extensive membrane surface, interacting more intensely with membrane-accumulated probes such as safranin, and larger matrix volumes to enhance the accumulation of intramitochondrial probes such as BCECF and PBFI. These differences in mitochondrial volume and membrane content were confirmed by using a flow cytometer to measure particle size (forward scattering) and structural complexity (side scattering) of isolated Bcl-2+ and Bcl-2− mitochondria (Fig. 4). The exact nature of the structural changes present in Bcl-2+ mitochondria is still not clear and will have to be investigated using three-dimensional imaging techniques since conventional electron microscopy does not show any striking differences between Bcl-2− and Bcl-2+ mitochondrial morphology (10Kowaltowski A.J. Vercesi A.E. Fiskum G. Cell Death Differ. 2000; 7: 903-910Crossref PubMed Scopus (140) Google Scholar). Independently of the exact nature of the structural alterations promoted by Bcl-2, our data using both fluorescent dyes and light scattering of individual mitochondria through flow cytometry clearly indicate that Bcl-2+ mitochondria are larger. The presence of larger mitochondria and, most probably, larger matrix volumes may explain why Bcl-2-overexpressing mitochondria have been previously found to present a larger capacity to accumulate Ca2+ ions (Ref. 26Murphy A.N. Bredesen D.E. Cortopassi G. Wang E. Fiskum G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9893-9898Crossref PubMed Scopus (386) Google Scholar, and see the scheme in Fig. 5) independently of their increased resistance to undergo non-selective inner membrane permeabilization following excessive Ca2+ uptake (mitochondrial permeability transition, Refs. 10Kowaltowski A.J. Vercesi A.E. Fiskum G. Cell Death Differ. 2000; 7: 903-910Crossref PubMed Scopus (140) Google Scholar and 11Kowaltowski A.J. Castilho R.F. Vercesi A.E. FEBS Lett. 2001; 495: 12-15Crossref PubMed Scopus (716) Google Scholar). It is also possible that the increased structural complexity of Bcl-2+ mitochondria is related to changes in membrane structure such as increases in cristal folds, resulting in resistance to cytochrome c loss under conditions in which the outer membrane is permeabilized (27Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar). Cytochrome c normally interacts closely with the inner membrane and must be displaced to the intermembrane space to be released into the cytosol (28Ott M. Robertson J.D. Gogvadze V. Zhivotovsky B. Orrenius S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1259-1263Crossref PubMed Scopus (796) Google Scholar). Finally, larger matrix volumes may explain why Bcl-2+ cells present higher quantities of matrix-soluble components such as NADPH, NADH, and glutathione (10Kowaltowski A.J. Vercesi A.E. Fiskum G. Cell Death Differ. 2000; 7: 903-910Crossref PubMed Scopus (140) Google Scholar, 14Esposti M.D. Hatzinisiriou I. McLennan H. Ralph S. J. Biol. Chem. 1999; 274: 29831-29837Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 23Ellerby L.M. Ellerby H.M. Park S.M. Holleran A.L. Murphy A.N. Fiskum G. Kane D.J. Testa M.P. Kayalar C. Bredesen D.E. J. Neurochem. 1996; 67: 1259-1267Crossref PubMed Scopus (201) Google Scholar). The antioxidant effects of NADPH and GSH are related to the increased resistance Bcl-2+ cells present to oxidative damage (10Kowaltowski A.J. Vercesi A.E. Fiskum G. Cell Death Differ. 2000; 7: 903-910Crossref PubMed Scopus (140) Google Scholar, 14Esposti M.D. Hatzinisiriou I. McLennan H. Ralph S. J. Biol. Chem. 1999; 274: 29831-29837Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 16Armstrong J.S. Jones D.P. FASEB J. 2002; 16: 1263-1265Crossref PubMed Scopus (206) Google Scholar, 23Ellerby L.M. Ellerby H.M. Park S.M. Holleran A.L. Murphy A.N. Fiskum G. Kane D.J. Testa M.P. Kayalar C. Bredesen D.E. J. Neurochem. 1996; 67: 1259-1267Crossref PubMed Scopus (201) Google Scholar). In summary, we found that Bcl-2 does not affect mitochondrial ΔΨ, ΔpH, or intramitochondrial K+ concentrations but alters mitochondrial structure, resulting in increased size and complexity. These changes are accompanied by an enhancement in the response to fluorescent dyes, including those that measure mitochondrial ΔΨ. Enhanced volume and structural complexity may affect the response presented by Bcl-2+ cells to normally deadly stimuli, inhibiting apoptosis and necrosis by preventing cytochrome crelease, increasing Ca2+ uptake capacity, and enhancing antioxidant defenses (see Fig. 5 for a proposed model). We acknowledge Edson Alves Gomes for excellent technical assistance and Prof. A. E. Vercesi for stimulating discussions and for allowing ready access to the flow cytometer. Dr. A. Starkov and Prof. E. J. Bechara are thanked for critical reading of the manuscript.
Referência(s)