Characterization of Mitochondrial and Extra-mitochondrial Oxygen Consuming Reactions in Human Hematopoietic Stem Cells
2005; Elsevier BV; Volume: 280; Issue: 28 Linguagem: Inglês
10.1074/jbc.m500047200
ISSN1083-351X
AutoresCláudia Piccoli, Roberto Ria, Rosella Scrima, Olga Cela, Annamaria D’Aprile, Domenico Boffoli, Franca Falzetti, Antonio Tabilio, Nazzareno Capitanio,
Tópico(s)Neutrophil, Myeloperoxidase and Oxidative Mechanisms
ResumoThis study was aimed to characterize the mitochondrial and extra-mitochondrial oxygen consuming reactions in human CD34+ hematopoietic stem cells. Cell samples were collected by apheresis following pre-conditioning by granulocyte colony-stimulating factor and isolated by anti-CD34 positive immunoselection. Polarographic analysis of the CN-sensitive endogenous cell respiration revealed a low mitochondrial oxygen consumption rate. Differential absorbance spectrometry on whole cell lysate and two-dimensional blue native-PAGE analysis of mitoplast proteins confirmed a low amount of mitochondrial respiratory chain complexes thus qualifying the hematopoietic stem cell as a poor oxidative phosphorylating cell type. Confocal microscopy imaging showed, however, that the intracellular content of mitochondria was not homogeneously distributed in the CD34+ hematopoietic stem cell sample displaying a clear inverse correlation of their density with the expression of the CD34 commitment marker. About half of the endogenous oxygen consumption was extra-mitochondrial and completely inhibitable by enzymatic scavengers of reactive oxygen species and by diphenylene iodinium. By spectral analysis, flow cytometry, reverse transcriptase-PCR, immunocytochemistry, and immunoprecipitation it was shown that the extra-mitochondrial oxygen consumption was contributed by the NOX2 and NOX4 isoforms of the O2−·. producer plasma membrane NAD(P)H oxidase with low constitutive activity. A model is proposed suggesting for the NAD(P)H oxidase a role of O2 sensor and/or ROS source serving as redox messengers in the activation of intracellular signaling pathways leading (or contributing) to mitochondriogenesis, cell survival, and differentiation in hematopoietic stem cells. This study was aimed to characterize the mitochondrial and extra-mitochondrial oxygen consuming reactions in human CD34+ hematopoietic stem cells. Cell samples were collected by apheresis following pre-conditioning by granulocyte colony-stimulating factor and isolated by anti-CD34 positive immunoselection. Polarographic analysis of the CN-sensitive endogenous cell respiration revealed a low mitochondrial oxygen consumption rate. Differential absorbance spectrometry on whole cell lysate and two-dimensional blue native-PAGE analysis of mitoplast proteins confirmed a low amount of mitochondrial respiratory chain complexes thus qualifying the hematopoietic stem cell as a poor oxidative phosphorylating cell type. Confocal microscopy imaging showed, however, that the intracellular content of mitochondria was not homogeneously distributed in the CD34+ hematopoietic stem cell sample displaying a clear inverse correlation of their density with the expression of the CD34 commitment marker. About half of the endogenous oxygen consumption was extra-mitochondrial and completely inhibitable by enzymatic scavengers of reactive oxygen species and by diphenylene iodinium. By spectral analysis, flow cytometry, reverse transcriptase-PCR, immunocytochemistry, and immunoprecipitation it was shown that the extra-mitochondrial oxygen consumption was contributed by the NOX2 and NOX4 isoforms of the O2−·. producer plasma membrane NAD(P)H oxidase with low constitutive activity. A model is proposed suggesting for the NAD(P)H oxidase a role of O2 sensor and/or ROS source serving as redox messengers in the activation of intracellular signaling pathways leading (or contributing) to mitochondriogenesis, cell survival, and differentiation in hematopoietic stem cells. Although hematopoietic stem cells (HSCs) 1The abbreviations used are: HSCs, hematapoietic stem cells; OX-PHOS, oxidative phosphorylation; ROS, reactive oxygen species; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine isothiocyanate; DPI, diphenylene iodinium; BICN, tert-butyl isocyanide; Me2SO, dimethyl sulfoxide; CCCP, carbonyl cyanide m-chlorophenylhydrazone; SOD, superoxide dismutase; NOX, NAD(P)H oxidase; H2DCF-DA, 2′,7′-dichlorodihydrofluorescein-diacetate; DCF, dichlorofluorescein; MDR1, multidrug resistant 1; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)-ethyl]glycine. 1The abbreviations used are: HSCs, hematapoietic stem cells; OX-PHOS, oxidative phosphorylation; ROS, reactive oxygen species; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine isothiocyanate; DPI, diphenylene iodinium; BICN, tert-butyl isocyanide; Me2SO, dimethyl sulfoxide; CCCP, carbonyl cyanide m-chlorophenylhydrazone; SOD, superoxide dismutase; NOX, NAD(P)H oxidase; H2DCF-DA, 2′,7′-dichlorodihydrofluorescein-diacetate; DCF, dichlorofluorescein; MDR1, multidrug resistant 1; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)-ethyl]glycine. have been gaining an extraordinary growing interest, in the last decades many aspects of their cellular biochemistry are still elusive and deserve attention (1To L.B. Haylock D.N. Simmons P.J. Juttner C.A. Blood. 1997; 89: 2233-2258Crossref PubMed Google Scholar). The main reason of such a gap in the knowledge of basic metabolic aspects of HSCs depends on the difficulties in obtaining enough, phenotypically pure, cell samples suitable for systematic functional biochemical analysis. Nevertheless, to acquire this information is of extreme importance, not only for the understanding of the mechanism underlying the processes leading to cell proliferation and differentiation, but also to improve and optimize the protocols for in vitro and in vivo cell recovery, maintenance and differentiation, applied in the cell therapy approaches where HSCs are believed to promise a profound change in the future of regenerative medicine (2Asahara T. Kalka C. Isner J.M. Gene Ther. 2000; 7: 451-457Crossref PubMed Scopus (163) Google Scholar, 3Alessandri G. Emanueli C. Madeddu P. Ann. N. Y. Acad. Sci. 2004; 1015: 271-284Crossref PubMed Scopus (53) Google Scholar). Adult HSCs reside in the bone marrow stroma where a very peculiar cellular and biochemical niche environment preserves them till appropriate stimuli activate their self-renewal and mobilization in the blood (4Cottler-Fox M.H. Lapidot T. Petit I. Kollet O. DiPersio J.F. Link D. Devine S. Hematology (Am. Soc. Haematol. Educ. Program). 2003; : 419-437Crossref Scopus (198) Google Scholar, 5Zhang J. Niu C. Ye L. Huang H. He X. Tong W.G. Ross J. Haug J. Johnson T. Feng J.Q. Harris S. Wiedemann L.M. Mishina Y. Li L. Nature. 2003; 425: 836-841Crossref PubMed Scopus (2370) Google Scholar, 6Pazianos G. Uqoezwa M. Reya T. BioTechniques. 2003; 35: 1240-1247Crossref PubMed Scopus (20) Google Scholar). Here they undergo a proliferative and multistage differentiation process leading to commitment toward all the blood cell lineages (7Leusch M.W. Daley G.O. Curr. Top. Dev. Biol. 2004; 60: 127-196Crossref PubMed Scopus (48) Google Scholar) and possibly other non-hematic cell types (8Heike T. Nakahata T. Int. J. Hematol. 2004; 79: 7-14Crossref PubMed Scopus (24) Google Scholar). The oxygen tension in the stromal niche, although difficult to assess experimentally, is thought to be extremely low (9Swartz H.M. Dunn J.F. Adv. Exp. Med. Biol. 2003; 530: 1-12Crossref PubMed Scopus (50) Google Scholar, 10Chow D.C. Wenning L.A. Miller W.M. Papoutsakis E.T. Biophys. J. 2001; 81: 675-684Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar), thus implying an almost anaerobic metabolic behavior whose low efficiency is well adapted to the low energy demand of this G0-G1 resting cell type. Furthermore, the hypoxic environment would limit the production of reactive oxygen species thus preserving from age-dependent oxidative damages such an essential cell reservoir. Upon mobilization of one of the two cell daughters, derived from the mother stem cell, in a normoxic milieu, a shift toward a more efficient aerobic metabolism is expected in view of the greater energetic effort required to sustain proliferation and differentiation. The larger availability of oxygen, experienced by the mobilized HSC, enables it to be used as the driving substrate for the mitochondrial oxidative phosphorylation as well as for other extra-mitochondrial oxidative reactions. In this work we have studied the enzymatic features of the cellular oxygen consuming reactions in human HSCs recovered from peripheral blood, upon mobilization from bone marrow by the growth factor granulocyte colony-stimulating factor (11Lapidot T. Petit I. Exp. Hematol. 2002; 30: 973-981Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar, 12Tabilio A. Falzetti F. Giannoni C. Aversa F. Martelli M.P. Rossetti M. Caputo P. Chionne F. Gambelunghe C. Martelli M.F. J. Hemathotherapy. 1997; 6: 227-234Crossref PubMed Scopus (37) Google Scholar, 13Fritsch G. Stimpfl M. Kurz M. Printz D. Buchinger P. Fischmeister G. Hoecker P. Gadner H. Bone Marrow Transplant. 1996; 17: 169-178PubMed Google Scholar, 14Bonnet D. J. Pathol. 2002; 197: 430-440Crossref PubMed Scopus (86) Google Scholar), and isolated by immunoselection using as cell specific tag the CD34 antigenic marker. Hematopoietic Stem Cells Source—HSCs, obtained upon informed consent from donors for allogeneic HSC transplant, were mobilized in the peripheral blood by recombinant granulocyte colony-stimulating factor treatment and collected by superparamagnetic iron-dextran particles directly conjugated to anti-CD34 as previously described (12Tabilio A. Falzetti F. Giannoni C. Aversa F. Martelli M.P. Rossetti M. Caputo P. Chionne F. Gambelunghe C. Martelli M.F. J. Hemathotherapy. 1997; 6: 227-234Crossref PubMed Scopus (37) Google Scholar, 15Aversa F. Tabilio A. Velardi A. Cunningham I. Terenzi A. Falzetti F. Ruggeri L. Barbabietola G. Aristei C. Latini P. Reisner Y. Martelli M.F. New Engl. J. Med. 1998; 339: 1186-1193Crossref PubMed Scopus (1017) Google Scholar). The purity of the isolated cells was evaluated by flow cytometry using a phycoerythrin-conjugated monoclonal anti-CD34 antibody and challenged against a large panel of antigens characterizing late committed or mature hematopoietic cell types as previously reported (12Tabilio A. Falzetti F. Giannoni C. Aversa F. Martelli M.P. Rossetti M. Caputo P. Chionne F. Gambelunghe C. Martelli M.F. J. Hemathotherapy. 1997; 6: 227-234Crossref PubMed Scopus (37) Google Scholar). After selection, aliquots of cells were freshly used or cryopreserved in liquid nitrogen. Before use the frozen cell samples were thawed at room temperature and washed twice in RPMI 1640 to remove the cryoprotectant Me2SO. Cell viability as determined by trypan blue exclusion was typically between 80 and 95%. Measurement of Oxygen Uptake—The rate of endogenous oxygen consumption was measured polarographically (the instrumental setting was computer-controlled) with a Clark-type oxygen electrode at 37 °C in 0.5 ml of RPMI 1640 without fetal bovine serum. After 10-15 min of baseline stabilization, 25 × 106 viable HSCs cells were added. Given the intrinsic low respiratory activity of the cell system a fine correction of the instrumental and/or medium-linked drift was absolutely necessary. A correction point by point for the first derivative of the non-linear drift, over the time course of the experiment, was applied. Comparable endogenous respiratory rate results were obtained with either freshly isolated and thawed samples. Spectrophotometric Analysis—5 × 106 cell in 100 μl of 100 mm Tris, pH 7.4, were lysed with 2% Triton X-100 in the presence of a protease inhibitor mixture. Optical spectra from 400 to 700 nm of the oxidized (by 10 μm ferricyanide) and reduced (by a few grains of dithionite) samples were recorded in a microvolume (50 μl) cuvette. The baseline drifts because of the residual turbidity of the suspension was largely removed by differential analysis (reduced minus oxidized) and further corrected for a polynomial baseline passing throughout the cytochromic isosbestic points (16Williams Jr., J.N. Arch. Biochem. Biophys. 1964; 107: 537-543Crossref PubMed Scopus (283) Google Scholar, 17North J.A. Rein D. Tappel A.L. Anal. Biochem. 1996; 233: 115-123Crossref PubMed Scopus (10) Google Scholar). Bidimensional Polyacrylamide Gel Electrophoresis—Samples of mitoplast proteins for blue native-PAGE were prepared as described by Schagger (18Schagger H. Methods Enzymol. 1995; 260: 190-202Crossref PubMed Scopus (132) Google Scholar). The first dimension native electrophoresis was run in a 5-12% acrylamide gradient. A lane was cut out of the first dimension gel and placed on a glass plate for incubation with lysis buffer at room temperature. The second dimension was a denaturing Tricine-SDS-PAGE. The proteins were visualized by silver staining. Densitometric analysis was performed by Versadoc imaging system, scanning the gel slabs along the lanes of the denaturating 2nd dimension run corresponding to the OXPHOS complexes separated in the 1st dimension run. Laser Scanning Confocal Microscopy—3 × 106/ml CD34+ HSCs were incubated with 500 nm MitoTracker green (30 min, 37 °C), washed with PBS, fixed with 3.7% paraformaldeyde (5 min, room temperature), resuspended in PBS plus 1% bovine serum albumin, and incubated with an anti-CD34 monoclonal antibody (1 h, room temperature). Then, HSCs were washed 3 times with PBS/bovine serum albumin, pH 7.4, and incubated (1 h, room temperature) with a secondary TRITC-conjugated antibody. The HepG2 cell line was treated with the mitochondrial probe as described for the HSCs and with TO-PRO 3 for nucleus staining (5 min, room temperature). Confocal microscopy was performed with a Leica TCSSP2 microscope. Fluorescent signals emitted by Mitotracker green (λex, 490 nm; λem, 516 nm) and by TRITC-conjugated secondary antibody (λex, 544 nm; λem 572 nm) were quantified by the Leica Confocal Software (LCS-TCS version 2.61). By means of the “stack” function for the defined area of the LCS-Analysis Tools it produced a xz intensity profile of the average value of the pixels within marked edges, including a single cell, as a function of each focal plane. Correction was made for the minimal background by repeating the procedure in a cell-free field. The integrated value of the xz profile was taken as a measure of the fluorescence intensity of that individual cell relative to the selected emission channel. About one hundred single cells were analyzed for both emission channels. Flow Cytometry—A Beckman Coulter Epics XL-MCL flow cytometer equipped with a 488-nm argon laser was used. To measure ROS production, samples were incubated with 10 μm 2′,7′-dichlorodihydrofluorescein-diacetate (H2DCF-DA) at 37 °C, protected from light for 30 min in the presence of 20 μm cyclosporin A. After loading with dye, the cells were washed by centrifugation (3 min at 300 × g) and resuspended in PBS. Samples contained 200,000 cells and 5,000 events for each sample were analyzed following the instrumental procedure (λem, 529 nm). Reverse Transcription-Polymerase Chain Reaction—3 μg of total cellular RNA isolated by TRIzol reagent was reverse transcribed to cDNA with specific antisense primers (50 pmol each, sequence shown in the legend of Fig. 3D) following the SuperScript Reverse Transcriptase protocol. Samples of 5 μl of reverse transcription reaction were PCR-amplified in a total volume of 50 μl with 50 pmol each of sense and antisense primers (sequences shown in the legend of Fig. 3D). The conditions were 35 cycles of denaturation at 94 °C (1 min), annealing at 60 °C (1 min), and extension at 72 °C (2 min); followed by a further 10-min extension. Purified PCR products were sequenced (3 times for each sample) on an automatic ABI Prism 310 DNA sequencer. Double Immunofluorescence Cytochemistry—HSCs were cytocentrifuged at 400 × g on polylysine-coated slides for 4 min, fixed (4% paraformaldehyde), permeabilized (0.2% Triton X-100), blocked (3% bovine serum albumin in PBS), and then sequentially incubated with the 1:200 diluted rabbit anti-gp91 and mouse anti-p47. After two washes in PBS/bovine serum albumin the sample was incubated with 10 μg/ml of the fluorescein 5-isothiocyanate-labeled sheep anti-rabbit IgG and Texas Red-labeled goat anti-mouse IgG. Fluorescence was evaluated with a Zeiss Axioplan 2 microscope. Immunoprecipitation and Immunoblotting—CD34+ HSC or polymorphonucleate cells (1 × 106) were lysed for 20 min in 1 ml of ice-cold lysis buffer (20 mm HEPES, pH 7.2, 150 mm NaCl, 1 mm EGTA, 10% glycerol, 1% Triton X-100, 1.5 mm MgCl2, 1 mm sodium vanadate, 2 mm sodium phosphate, and protease inhibitors mixture). Lysates were centrifuged at 12,000 rpm for 15 min and the supernatants incubated (2 h, 4 °C) with rabbit preimmune serum and 50 μl of a 50% Protein G-Sepharose slurry for preclearing, the after spinning supernatant was then incubated overnight at 4 °C with rabbit anti-gp91 (Upstate or Santa Cruz), anti-p67 (Upstate), or goat anti-p47 (Santa Cruz) polyclonal antibody and Protein A-Sepharose beads. Immunoprecipitates were washed in HNTG buffer, suspended in Laemmli's buffer, and run on 8% SDS-PAGE followed by Western blot. gp91phox, p67, p47, and serine-phosphorylated peptides were immunodetected by enhanced chemiluminescence under not limiting detecting antibodies. Statistical Analysis—Two tailed Student's t test was applied to evaluate the significance of differences measured throughout the data sets reported. Mitochondrial Oxygen Consumption in HSCs—Fig. 1 clusters the results of a multifaceted approach aimed to characterize the mitochondrial oxidative phosphorylation (OXPHOS) system in peripheral blood granulocyte colony-stimulating factor-mobilized CD34+ HSCs. Fig. 1, panel A, shows the outcome of a systematic high-resolution respirometric analysis, carried out on dense cell suspensions. The rate of endogenous cell respiration resulted on an average basis to be about 84 pmol of O2 consumed per min/106 cells (10-15 different preparations of HSC). This overall oxygen consumption rate was 60% inhibited by the cytochrome c oxidase inhibitor cyanide. Addition of antimycin A plus myxothiazol (inhibitors of cytochrome c reductase) resulted in a similar extent of inhibition of the respiratory activity (data not shown). Thus the measured respiratory activity attributable to mitochondria amounted to about 50 pmol of O2/106 cells/min. This value, when compared with that of other cell types measured under identical conditions, resulted in being at least 10 times lower (Ref. 19Villani G. Greco M. Papa S. Attardi G. J. Biol. Chem. 1998; 273: 31829-31836Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar and confirmed by ourselves). The CN-sensitive endogenous respiration was not enhanced by the addition of the protonophore un-coupler CCCP and was slowed down by the ATP-synthase inhibitor oligomycin (about 40% inhibition), indicating that the measured mitochondrial oxygen consumption rate featured an active (phosphorylating) state. The low endogeneous respiratory control ratio attained was likely because of the intrinsically limited activity of the cell sample. It must be mentioned that the experiments reported were carried out with cells obtained upon thawing of the frozen samples and immediately assayed soon after removal of Me2SO, used to preserve cell viability upon freeze-thaw. The cell viability tested by the trypan blue exclusion assay was never below 95%. Comparable results were obtained with fresh cell samples assayed a few hours after collection. The correlation between mitochondrial respiratory activity and cytochrome content in HSCs was assessed by differential spectrophotometric analysis on cell lysate. To avoid any loss of the overall cytochrome content, we preferred to carry out analysis on the whole detergent-solubilized cell suspension without separation of the particulate by centrifugation. Fig. 1, panel B, shows the absorbance difference visible spectra (dithionite reduced minus air oxidized) of whole cell lysate obtained upon Triton X-100 treatment (averaged from different HSCs preparation and normalized to 1 × 106 cells/ml). The spectra clearly indicated the presence of canonical mitochondrial a, b, and c type cytochromes both in the α-β (500-650 nm) and the Soret (400-500 nm) regions (16Williams Jr., J.N. Arch. Biochem. Biophys. 1964; 107: 537-543Crossref PubMed Scopus (283) Google Scholar, 17North J.A. Rein D. Tappel A.L. Anal. Biochem. 1996; 233: 115-123Crossref PubMed Scopus (10) Google Scholar). A quantitative analysis, expressed as cytochrome c oxidase content (whose absorption at 605 nm is scarcely affected by optical overlapping because of the presence of contaminating chromophores), resulted in a value of 0.3 pmol/106 cells, which was again relatively much lower than that reported and confirmed by us in other cell types. However, when the measured CN-sensitive respiratory activity was referred to the cytochrome oxidase content (which is considered to be the main rate-limiting step for mitochondrial respiration under in vivo conditions (19Villani G. Greco M. Papa S. Attardi G. J. Biol. Chem. 1998; 273: 31829-31836Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar)) a specific turn-over number of about 10 e-/aa3/s was calculated, which is comparable with the specific activity reported under steady-state respiration for other cell types (19Villani G. Greco M. Papa S. Attardi G. J. Biol. Chem. 1998; 273: 31829-31836Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). Measurement of the oxygen consumption rate in the presence of nitro-l-arginine methylester, an inhibitor of the NO synthase (whose product is known to result, under certain conditions, in inhibition of cytochrome c oxidase activity (20Brunori M. Giuffre A. Forte E. Mastronicola D. Barone M.C. Sarti P. Biochim. Biophys. Acta. 2004; 1655: 365-371Crossref PubMed Scopus (89) Google Scholar)), did not result in any appreciable increase of respiration (data not shown). Thus the low mitochondrial respiratory activity measured in our HSC samples was because of a relatively poor content of mitochondrial cytochromes rather than to an inhibitory control. This conclusion was further supported detecting by two-dimensional blue native-SDS-PAGE of mitoplast proteins with the pattern of subunits constituting the mitochondrial respiratory complexes (I to IV) as well as the ATP-synthase complex (Fig. 1, panel C). It can be noted that, although the overall composition of the OXPHOS complex subunits was comparable with that of mitoplasts prepared from HepG2 (taken as reference for a mitochondria-rich cell type), their content resulted in being lower. A densitometric analysis of the subunits assembled in OXPHOS complexes I to V confirmed that in mitoplasts from HSC CD34+ there was roughly half of the amount of that present in HepG2 (Fig. 1C, bottom panel). Considering that the comparison was made between the densitometric profiles normalized to the amount of mitoplast proteins loaded on the gel, it would indicate that the observed poor content of components of the mitochondrial OXPHOS system in HSCs was because of their lower density per mitochondria rather than (or in addition) to a lower amount of mitochondria per cell unit. To address this issue, the intracellular mitochondrial content was directly pinpointed in HSCs by laser scanner confocal microscopy analysis using MitoTracker green (a fluorochrome specifically accumulating in membrane potential-generating mitochondria). In addition, the same HSC sample was treated with a fluorophore-conjugated antibody recognizing the CD34 antigen marker. A typical outcome of such an analysis is shown in Fig. 2A from which a number of indications could be drawn. The morphology of the mitochondrial network resulted in most of the HSCs in a bipolar perinuclear clustering differently from the more intense and widespread appearance of the mitochondrial signal in the cytoplasm of HepG2. A diverse interorganelle connectivity has been recently described to be dependent on the cell-cycle phase (21Capaldi R.A. Aggeler R. Gilkerson R. Hanson G. Knowles M. Marcus A. Margineantu D. Marusich M. Murray J. Oglesbee D. Remington S.J. Rossignol R. Biochim. Biophys. Acta. 2002; 1555: 192-195Crossref PubMed Scopus (24) Google Scholar). The density of the intracellular mitochondrial content was, however, not similar throughout the HSC sample. Some cell types showed, indeed, a faint staining with MitoTracker, whereas others displayed a much higher ability to load the mitochondrial probe (identical results were obtained with 3 different preparations of HSCs). Similarly the CD34 cell surface marker immunodetected in the same sample exhibited also a highly variable intercellular degree of clustered labeling. Noteworthy, the higher the CD34 signal density in a given HSC the lower the mitochondrial content in it, and vice versa. A quantitative estimation of the relative fluorescence signals, carried out on a large number of single cell imaging, showed a clear inverse correlation between CD34 and mitochondria content (Fig. 2B). Extra-mitochondrial Oxygen Consumption in HSCs—The relative large CN-insensitive oxygen consumption rate exhibited by the HSC samples prompted us to verify the possible involvement of other cellular oxygen consuming reactions in addition to that elicited by the mitochondrial cytochrome c oxidase converting dioxygen to water molecules. First we tested the effect of externally added enzymatic scavengers of reactive oxygen species. Fig. 3A shows that addition of superoxide dismutase together with catalase strongly reduced the endogenous oxygen consumption rate by about 50%. Successive addition of cyanide further inhibited the respiratory activity, leaving a residual activity amounting to about 10% of the initial endogenous respiration. When the order of the additions was inverted, the same result was obtained and when SOD plus catalase were tested in the absence of cells no detectable effect was observed on the instrumental O2 consumption drift (not shown). This result indicated the occurrence of partial reduction of dioxygen to the superoxide anion and/or hydrogen peroxide revealed by the concerted action of the two antioxidant enzymes, which regenerated dioxygen. The relative large amount of ROS produced could not be ascribed to electron leaks of the mitochondrial respiratory chain activity because this has been reported never to exceed 2-3% of the O2 consumed under the controlled state IV condition and to be even lower under the active phosphorylating regimen (23Cadenas E. Davies K.J. Free Radic. Biol. Med. 2000; 29: 222-230Crossref PubMed Scopus (2349) Google Scholar). Moreover as the effect of the two antioxidant enzymes was exerted outside the cell, it seemed unlikely that intracellular ROS production could escape the endogenous antioxidant battery, rather suggesting a periplasmic location of the ROS generating system(s). A reexamination of the differential spectra of the whole HSC lysate (shown in Fig. 1B) compared with that of isolated mitochondria (not shown) revealed in the former a relatively larger amount of cytochromes absorbing in the 550-560 and 420-430 nm regions, when related to the absorbance of cytochrome c oxidase. This suggested the presence of a significant amount of extra-mitochondrial cytochromes (see below). In addition, the differential absorbance at 470-475 nm clearly indicated the presence of myeloperoxidase (which specifically absorbs in that region (24Floris R. Kim Y. Babcock G.T. Wever R. Eur. J. Biochem. 1994; 222: 677-685Crossref PubMed Scopus (7) Google Scholar)). It is known that in many cell types the major ROS producer (even more efficient, under certain conditions, than the mitochondrial respiratory chain) is the cell membrane-bound NAD(P)H oxidase (NOX) (25Babior B.M. Blood. 1999; 93: 1464-1476Crossref PubMed Google Scholar), which oxidizes NAD(P)H, transferring electrons to dioxygens, which is partially reduced to the superoxide anion (26Cross A.R. Segal A.W. Biochim. Biophys. Acta. 2004; 1657: 1-22Crossref PubMed Scopus (363) Google Scholar). Being a flavoenzyme, NOX is efficiently inhibited by diphenylene iodinium (DPI) (27Hancock J.T. Jones O.T. Biochem. J. 1987; 242: 103-107Crossref PubMed Scopus (193) Google Scholar). To verify this possibility we tested the effect of DPI on the endogenous respiration. As shown in Fig. 3A, DPI, at a concentration reported to inhibit NOX, resulted in a marked decrease of the endogenous respiratory activity (by about 50%). Successive addition of cyanide completely inhibited the DPI-insensitive oxygen consumption rate. When the order of addition of the two inhibitors was inverted, complementary results were observed and, more important, addition of SOD plus catalase after inhibition by DPI did not cause any effect on the oxygen consumption rate (data not shown). Direct measurement of cellular ROS production was assessed by flow cytometry using DCF-DA as a specific probe for H2O2 detection. DCF-DA permeates cell membranes, is deacetylated by intracellular esterase, and becomes fluorescent upon oxidation. Although DCF is membrane impermeant and thus should remain trapped inside the cell it can also be the substrate of the P-glycoprotein multidrug resistant (MDR1) pump (28Bernardi P. Petronilli V. Di Lisa F. Forte M. Trends Biochem. Sci. 2001; 26: 112-117Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar). Thus cells expressing MDR1 can slowly release DCF unless treated with MDR1 inhibitors. Because it is long known that HSCs express significant amounts of MDR1 (29Drach D. Zhao S. Drach J. Mahadevia R. Gattringer C. Huber H. Andreeff M. Blood. 1992; 80: 2729-2734Crossref PubMed Google Scholar), during the HSC assay we treated HSCs with a MDR1 inhibitor. Fig. 3B shows that incubation of HSC with cyclosporin A (a specific inhibitor of MDR) (28Bernardi P. Petronilli V. Di Lisa F. Forte M. Trends Biochem. Sci. 2001; 26: 112-117Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar) allowed detection of production of intracellular ROS and tha
Referência(s)