AJP - Regu Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Regul Integr Comp Physiol 289: R1756-R1762, 2005; doi:10.1152/ajpregu.00480.2004
0363-6119/05 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (8)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Therade-Matharan, S.
Right arrow Articles by Vicaut, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Therade-Matharan, S.
Right arrow Articles by Vicaut, E.

ENVIRONMENTAL, EXERCISE AND RESPIRATORY PHYSIOLOGY

Reactive oxygen species production by mitochondria in endothelial cells exposed to reoxygenation after hypoxia and glucose depletion is mediated by ceramide

S. Therade-Matharan,1 E. Laemmel,1 S. Carpentier,2 Y. Obata,1 T. Levade,2 J. Duranteau,3 and E. Vicaut1

1Laboratoire d'Etude de la Microcirculation, Equipe d’accueil, Faculté de Médecine Paris, France; 2Institut National de la Santé et de la Recherche Médicale, Laboratoire de Biochimie Centre Hospitalier Universitaire Rangueil, Toulouse, France; and 3Equipe d’accueil, Laboratoire d'Anesthésie, Hôpital de Bicêtre, Kremlin Bicêtre, France

Submitted 19 July 2004 ; accepted in final form 2 August 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In endothelium, reoxygenation after hypoxia (H/R) has been shown to induce production of reactive oxygen species (ROS) by complex III of the mitochondrial respiratory chain. The purpose of the present study was to test the involvement of ceramide in this phenomenon. Human umbilical vein endothelial cells underwent 2 h of hypoxia (PO2, ~20 mmHg) without glucose and 1 h of reoxygenation (PO2, ~120 mmHg) with glucose. ROS production was measured by the fluorescent marker 2',7'-dichlorodihydrofluorescein diacetate, and cell death by propidium iodide. We showed that 1) after 1 h of reoxygenation, fluorescence had risen and that ROS production was inhibited by desipramine, an inhibitor of sphingomyelinase, an enzyme responsible for ceramide production (126 ± 7% vs. 48 ± 12%, P < 0.05); 2) administration of ceramide (N-acetylsphingosine) per se (i.e., in the absence of H/R) induced ROS production (65 ± 3%), which was inhibited by complex III inhibitor: antimycin A (24 ± 3%, P < 0.0001), or stigmatellin (31 ± 2%, P < 0.0001); 3) hypoxia/reoxygenation-induced ROS production was not affected by either ceramide-activated protein kinase inhibitor dimethyl aminopurine or mitochondrial permeability transition inhibitor cyclosporin A but was significantly inhibited by the antiapoptotic protein Bcl-2 (82 ± 8%, P < 0.05); 4) ceramide-induced ROS production was also inhibited by Bcl-2 (41 ± 4%, P < 0.0001). These results demonstrate that in endothelial cells submitted to hypoxia and glucose depletion followed by reoxygenation with glucose, the pathway implicated in mitochondrial complex III ROS production is ceramide dependent and is decreased by the antiapoptotic protein Bcl-2.

H2O2

ALTHOUGH HYPOXIA ALONE CAN produce tissue injury, different types of evidence suggest that a burst of reactive oxygen species (ROS) generation coincides with reoxygenation. The evidence that ROS are essential mediators of hypoxia/reoxygenation (H/R) injury is based on the observation that various chemical and enzymatic antioxidants appear to protect against the tissue damage seen with reperfusion (16, 17). ROS gener-ated by hypoxia or reoxygenation are now recognized as interacting with physiological signal transducers rather than behaving like simple reactants that peroxidize membrane lipids, oxidize DNA, or denature enzyme proteins. Because endothelial cells are ubiquitous and located at the blood-brain barrier, they have been proposed as the central source of ROS burst that occurs in ischemic tissues on reoxygenation (47, 59).

In a previous study (50), we showed that the main site of production of ROS in human umbilical vein endothelial cells (HUVEC) after hypoxia/reoxygenation (H/R) was the cytochrome (cyt) b of the complex III of the mitchondrial electron transport chain. In several types of cells, ceramide issued from activation of a sphingomyelinase that cleaves membrane sphingomyelin (SM) has been demonstrated to be involved in tissue injury after ischemia or anoxia (22, 32, 46, 55, 58). In isolated mitochondria (17) or in L929 cells (43), ceramide has been shown to interfere with several mitochondrial mechanisms, including cyt c release, which compromises mitochondrial electron flow and triggers ROS formation. In addition, in a previous study (9), we showed that ceramide was involved in the mitochondrial ROS production by HUVEC exposed to TNF-{alpha}.

Interactions between ceramide and mitochondria have been proposed to be either direct (10, 14, 19) or indirect. Regarding the latter, several mechanisms have been reported, such as the activation of protein kinase (9, 31), the induction of the mitochondrial permeability transition (MPT) (38, 39), and the modulation of the phosphorylation of the antiapoptotic protein Bcl-2 (27, 42, 53).

The present study attempts to elucidate the role of ceramide in H/R-induced ROS formation. For this purpose, the following approach was used: 1) to demonstrate that sphingomyelinase that hydrolyzed sphingomyelin to form ceramide was involved in ROS production in endothelial cells submitted to H/R, 2) to demonstrate that ceramide can directly induce ROS production by a mechanism similar to that found after H/R, and 3) to test the possibility that the H/R-induced ROS formation was either related to the activation of a protein kinase, required the induction of MPT, or was sensitive to Bcl-2.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and culture. HUVEC were obtained from PromoCell Laboratories (Heidelberg, The Netherlands) as cryopreserved cells. After thawing, cells were plated (5,000 to 10,000 cells/cm2) in culture flasks precoated with rat-tail type I collagen (5 g/cm2, Böhringer-Mannheim, Mannheim, Germany) and cultured to confluence in endothelial cell growth medium containing 2% fetal calf serum, 0.1 ng/ml human recombinant epidermal growth factor, 1.0 ng/ml human recombinant basic fibroblast growth factor, 1.0 µg/ml synthetic hydrocortisone, 50 µg/ml gentamicin sulfate, 50 ng/ml amphotericin B, and 0.62 ng/ml phenol red. Medium was supplemented with Supplement Mix (PromoCell) containing endothelial cell growth factor and heparin and with 1% penicillin-streptomycin solution (Sigma, St. Louis, MO). A cell suspension in nutritive medium was plated onto plastic Thermanox coverslips 25-mm diameter (Poly Labo, Strasbourg, France) to obtain a seeding density of ~5 x 103 cells/cm2. The medium was replaced every 48 h, and cells were maintained in a humidified air with 5% CO2 atmosphere at 37°C until confluence was reached. All cells were studied at the third passage.

Perfusion system. Endothelial cell monolayers on plastic coverslips were placed in a stainless steel flow-through chamber (volume, 1 ml; Penn Century, Philadelphia, PA). The chamber was sealed using thin wafer gaskets and mounted in a heated (37°C) water serpentine on an inverted microscope (Leica SA, Rueil-Malmaison, France). The perfusate consisted of Krebs-Henseleit bicarbonate buffer (pH 7.4, 37°C) containing (in mM): 117.3 NaCl, 4.7 KCl, 25 NaHCO3, 1.3 MgSO4, 1.2 KH2PO3, 1.23 CaCl2, 1 lactate, and 11.1 glucose; the buffer was equilibrated during stabilization and reperfusion in an atmosphere of 20% O2-5% CO2-75% N2. In the perfusion chamber, PO2 was ~120 mmHg and the perfusate pH was 7.40. During hypoxia, the perfusate was equilibrated with 5% CO2-95% N2 (PO2, ~20 mmHg in the chamber and pH 7.38). The perfusion system was a nonrecirculating system with a constant flow rate of 0.5 ml/min.

Fluorescence and light microscopy. An inverted microscope (model DM-IRB; Leica) was equipped for epifluorescent illumination and included a mercury light source (50 W), a 12-bit digital cooled camera (model RTE/CCD-1317-K/1; Princeton Instruments, Trenton, NJ), and a shutter (model D-122; Uniblitz, Rochester, NY) under computer control (Metamorph Imaging System 3.5, Universal Imaging, West Chester, PA), with appropriate excitation and emission cubes. Fluoresent cell images were obtained using a x20 objective for fluorescence (Leica). Data were acquired and analyzed using Metamorph software (Universal Imaging).

Measurement of ROS and assessment of cell death. ROS generation in HUVEC was assessed using the 2',7'-dichlorofluorescein (DCF) probe (Sigma, St. Louis, MO), which is very sensitive probe to ROS (H2O2 and OH–) (51). The reduced membrane-permeable form of the dye H2DCF-DA was added to the perfusate at a final concentration of 5 µM. Within the cell, esterases cleave the acetate groups on H2DCF, thus trapping the H2DCF intracellularly. ROS in the cells oxidizes H2DCF, yielding the fluorescent product DCFH (33, 44). After 1 h, fluorescence was measured every 10 min using an excitation wavelength of 480 ± 20 nm, a dichroic 505-nm-long pass, and an emitter bandpass of 527 ± 15 nm. Previous studies of the behavior of DCFH revealed that the DCFH probe is readily oxidized by H2O2 or hydroxyl radicals but is relatively insensitive to superoxide (40, 51).

To evaluate cell viability, we used the fluorescent probe propidium iodide (PI; Sigma), which was added to the perfusate at a final concentration of 5 µM. This probe is a red fluorescent, cell-impermeant dye that is widely used to detect dead or dying cells and only penetrates into the cell when the membrane is injured and binds to the DNA. Once this dye is bound to nucleic acids, its fluorescence is enhanced 20- to 30-fold, its excitation maximum shifts ~30 to 40 nm into the red, and its emission maximum ~15 nm into the blue. After 1 h of perfusion, fluorescence was measured every 30 min by using an excitation wavelength of 515–560 nm, a dichroic 580-nm long pass, and an emitter bandpass of 590 nm.

Fluorescence intensity was assessed by the imaging system Metamorph 3.5 over a region of interest that included the whole observational field. Intensity values are reported as percentage changes of initial values.

Experimental protocol. Cells plated onto plastic coverslips were placed in the perfusion chamber and flow was started. HUVEC cells subjected to H/R underwent 1 h of stabilization in Krebs-Henseleit buffer under normoxic conditions with glucose, and were then exposed for 2 h to hypoxia without glucose and with deoxy-D-glucose blocking glycolysis in the perfusion chamber. Perfusion was then returned for 1 h under normoxic conditions with glucose. Control HUVEC underwent 1 h of stabilization, followed by exposure for 3 h to the same Krebs-Henseleit buffer (20% O2-5% CO2-75% N2) with glucose but without H/R. HUVEC cells subjected to ceramide (C2) underwent 1 h of stabilization in Krebs-Henseleit buffer under normoxic conditions and was then exposed for 1 h to ceramide in the perfusion chamber. Control HUVEC underwent 1 h of stabilization, followed by exposure for 1 h to the same Krebs-Henseleit buffer (20% O2-5% CO2-75% N2) without ceramide. During these experimentations, one fluorescent image of H/R or ceramide and control cells was acquired every 10 min and analyzed to assess ROS production, and one image was acquired every 30 min and analyzed to assess cell death.

To test the involvement of the acidic sphingomyelinase (ASMase), which hydrolyzed sphingomyelin, cell responses to H/R were studied in the presence of the ASMase inhibitor desipramine (5 µM) (9, 52).

Then, to ascertain more directly the effect of ceramide, we studied ROS production in cells submitted to superfusion of Krebs + ceramide (N-acylsphingosine, C2, 20 µM) (17) during 1 h. To test the hypothesis that the effect of ceramide on ROS production in cells involved complex III, as previously shown for H/R (50), the same experiments were carried out in the presence of two different complex III inhibitors: antimycin A (AA; 10 µM) (8), a quinone analog that binds to cyt b562 and blocks electron transport to the ubiquinone in complex III, and stigmatellin (stig; 10 µM) (1), which blocks electron transport to both the "Rieske" [2Fe-2S] iron sulfur protein and heme b566 (18, 25). Both inhibitors were administered 30 min before and during ceramide administration.

To elucidate the mechanisms by which H/R ceramide-dependent pathway may affect mitochondrial ROS production, we studied the response to H/R in the presence of either the ceramide-activated protein kinase (CAPK) inhibitor dimethylaminopurine (DMAP; 1 mM) (31), or the mitochondrial permeability transition (MPT) inhibitor, cyclosporin A (CsA; 2 µM) (34) or the antiapoptotic protein Bcl-2 (40 nM) (17), which is known to be located at mitochondrial outer membrane and to inhibit cyt c release (6, 17, 27, 53).

In a complementary series of experiments, we checked that the effect we observed with Bcl-2 on H/R-induced ROS production was also found on ceramide-induced ROS production. For this purpose, cells were submitted to superfusion of Krebs + ceramide (N-acylsphingosine, C2, 20 µM) in the presence of Bcl-2 (40 nM) during 1 h. The inhibitor was administered 30 min before and during ceramide administration.

Finally, using dosages of ceramide we confirmed directly that ceramide levels increased during H/R. For this purpose, we submitted HUVEC to a sequence of H/R similar to that used for ROS measurements (n = 7 at each time) and compared it to control HUVEC under normoxic conditions for the same period of time (n = 4 at each time). For measurements, we used the methods as described by Bielawska et al. (3) and Malagarie-Cazenave et al. (30). The lipids were extracted with chloroform/methanol from the HUVEC lysates prepared for DEVDase assay. The ceramide content was determined using Escherichia coli diacylglycerol kinase (kindly provided by Drs. D. Perry and Y. A. Hannun, Charleston, SC) and [_-32P]ATP (6,000 Ci/mmol; PerkinElmer Life Sciences).

Presentation of the results and statistical analysis. Differences between groups were analyzed using one-way or two-way ANOVA (within factor time between factor experimental vs. control). Statistical significance was set at the 0.05 level. Values are reported as means ± SE.

Reagents. All products, reagents, and inhibitors were purchased from Sigma-Aldrich, as specified above. Bcl-2 is from Sigma-Aldrich; the protein sequence is truncated at the COOH-terminal sequence.

Inhibitors were dissolved in DMSO. To avoid repeated freezing and thawing, working aliquots were prepared and stored at –20°C.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of sphingomyelinase inhibitor on ROS production during exposure to H/R. After 2 h of hypoxia, during which the rise in ROS production was nil, reoxygenation induced a rapid production of ROS in HUVEC. This was evidenced by the significant increase in DCF fluorescence, which reached 126 ± 7% after 1 h of reoxygenation (fig. 1). In contrast, this production was low (14 ± 2%) in control cells (P < 0.0001).



View larger version (11K):
[in this window]
[in a new window]
  		Fig. 1. Effect of reoxygenation after hypoxia (H/R) on reactive oxygen species (ROS) production and effect of sphingomyelinase inhibitor on ROS production during exposure to H/R. Data are presented as means ± SE and represent the difference between the last hypoxic point and the last reoxygenation point for the H/R group and the desipramine (Desip) group when cells are incubated in the presence of the acidic sphingomyelinase (ASMase) inhibitor Desip (5 µM, n = 7). The difference between the measurements was made at the corresponding times in the control group. *A significant rise in fluorescence was observed in cells submitted to H/R (n = 8) compared with the control cells (P < 0.0001, n = 6). ROS production was significantly decreased by Desip compared with the H/R group (P < 0.05).

 
As shown in Fig. 1, a 3-h incubation of HUVEC during H/R with ASMase inhibitor desipramine (Desip; 5 µM) inhibited ROS production by ~62%. Indeed, the value of ROS increase fell to 48 ± 12% in the presence of desipramine (P < 0.05 compared with H/R group, n = 7).

Effect of H/R on ceramide content. As shown in Fig. 2, we confirmed the involvement of ceramide in the effect of H/R, because the dosage of ceramide content clearly showed that an increase in ceramide was induced during H/R (P < 0.05). Note, however, that the increase in ceramide began during the hypoxic phase of the experiments.



View larger version (14K):
[in this window]
[in a new window]
  		Fig. 2. Effect of H/R on ceramide formation. Data are presented as means ± SE. We submitted human umbilical vein endothelial cells (HUVEC) to a sequence of H/R similar to that used for ROS measurements (n = 7 at each time) and compared it to control HUVEC under normoxic conditions for the same period of time (n = 4 at each time). Ceramide levels increased significantly (P < 0.05) at each time of the experiments.

 
ROS production induced by ceramide and the effect of complex III inhibitors. As shown in Fig. 3, when HUVEC were incubated with ceramide (C2), ROS production increased to 65 ± 3% after 1 h of exposure. When HUVEC were incubated with C2 and the complex III inhibitors AA or Stig, ROS production after a 1-h exposure was significantly inhibited (24 ± 3%, P < 0.0001, and 31 ± 2%, P < 0.0001, respectively, n = 6 in each case). In control experiments, we have checked that these inhibitors did not modify fluorescence by an unspecific manner.



View larger version (12K):
[in this window]
[in a new window]
  		Fig. 3. Effect of complex III inhibitors on ROS production in the presence of ceramide. Cells were incubated in the presence of Krebs (Ctl), or ceramide (C2), or C2 associated with either the complex III inhibitor antimycin A (AA, 10 µM, n = 6), which inhibits cytochrome b or stigmatellin (Stig, 10 µM, n = 6), which inhibits electron transfer between ubiquinol and FeS cluster. Data are presented as means ± SE and represent the difference between the point of the beginning of exposure to the inhibitor and the point of the end of the experiment (90 min after the beginning). *ROS production was largely inhibited by AA (P < 0.0001) and Stig (P < 0.0001) compared with the C2 group.

 
Effect of different inhibitors of signaling pathway on ROS production. As shown in Fig. 4, inhibition of CAPK by DMAP did not inhibit H/R-induced ROS production. Similarly inhibition of mitochondrial permeability transition by CsA did not inhibit H/R-induced ROS production. In contrast, a significant inhibition was found with Bcl-2 (82 ± 8%) (P < 0.05, n = 6).



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 4. Effects of signaling pathway inhibitors on ROS production in HUVEC exposed to H/R. Data are presented as means ± SE and represent the difference between the last hypoxic point and the last reoxygenation point when cells are exposed to H/R and incubated in the presence of the ceramide-activated protein kinase (CAPK) inhibitor dimethylaminopurine (DMAP; 1 mM, n = 6), the mitochondrial permeability transition (MPT) inhibitor cyclosporine A (CsA; 2µM, n = 6) and the antiapoptotic protein Bcl-2 (40 nM, n = 6). In control groups (Ctl; n = 6), cells underwent the same total duration of experiment without any changes in superfusate. *ROS production in the control group was significantly lower than in H/R group (P < 0.0001) and was significantly decreased by Bcl-2 compared with H/R group (P < 0.05). No significant effect was found with the other inhibitors. DCF, 2',7'-dichlorofluorescein.

 
In addition, as shown in Fig. 5, we also found in complementary experiments (n = 3) that Bcl-2 also inhibited ceramide-induced ROS production (65 ± 3% vs. 41 ± 4%, P < 0.0001).



View larger version (11K):
[in this window]
[in a new window]
  		Fig. 5. Effect of antiapoptotic protein Bcl-2 on ROS production in the presence of ceramide. Cells were incubated in the presence of Krebs (Ctl), or ceramide (C2), or C2 associated with antiapoptotic protein Bcl-2 (40 nM, n = 3). Data are presented as means ± SE and represent the difference between the point of the beginning of exposure to the inhibitor and the point of the end of the experiment (90 min after the beginning). *ROS production was inhibited by Bcl-2 (P < 0.0001) compared with ceramide group.

 
Cell death. PI fluorescence, which reflects cell death, did not vary significantly in HUVEC exposed to H/R or in control cells. When studying the different inhibitors that have been previously tested for their possible effect on ROS production, we did not find any significant change in PI fluorescence compared with H/R exposed HUVEC not treated with inhibitors (Fig. 6A).



View larger version (13K):
[in this window]
[in a new window]
  		Fig. 6. Effects of signaling pathway inhibitors during H/R and complex III inhibitors with C2 on cellular death. A: cells were incubated in the presence of the ASMase inhibitor desipramine (Desip, 5 µM, n = 7), the CAPK inhibitor DMAP (1 mM, n = 5), the MPT inhibitor cyclosporine A (CsA, 2 µM, n = 7), the antiapoptotic protein Bcl-2 (40 nM, n = 6). B: cells were incubated in the presence of N-acyl-sphingosine alone (C2, 20 µM, n = 6), and associated with antimycin A (AA, 10 µM, n = 6) or stigmatellin (Stig, 10 µM, n = 6). These inhibitors were tested for their effects on PI fluorescence in HUVEC exposed to H/R or ceramide (C2). Data are presented as means ± SE and represent the difference between the first point and the last point of the experiment. No significant effect was found between H/R group and other groups or between C2 group and C2+AA or C2+Stig.

 
We also found that ceramide + complex III inhibitors did not significantly change PI fluorescence either compared with ceramide alone or to control HUVEC (Fig. 6B).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study was designed to identify the signaling pathway of ROS production in endothelial cells submitted to conditions close to those occurring during hemorrhagic shock.

Because endothelial cells are an important source of ROS, we used HUVEC, which are commonly used as an in vitro model to study hypoxia-reoxygenation (48, 60). HUVEC were submitted to 2 h of hypoxia, because Zweier et al. (60) showed that 90 min of anoxia is the minimal time required for large ROS production during reoxygenation.

In addition, it should be stressed that when hypoxia is due to hemodynamic perturbations, the nutrient supply can also be severely diminished. Thus, in the present study, we associated hypoxia at a level very close to that found in terminal vessels during hemorrhagic shock (35, 48) and glucose depletion, which induce a relevant additional stress (29, 36), and we associated the subsequent reoxygenation with glucose reintroduction into the perfusate. Note, however, that in additional control experiments (data not shown), we checked that glucose depletion was not the determinant for explaining our finding, as we observed similar H/R-induced increases in ROS even in the presence of normal glucose. In this experimental model, we used fluorescence microscopy to quantify ROS production. Recent results in this model provided evidence that in endothelial cells, a major site of H/R-induced ROS production is the cyt b of complex III of mitochondrial respiratory chain (50). However, the signaling pathways of H/R-induced ROS production have not yet been described. The present study was carried out to know whether ceramide is a mediator in this ROS production and what are the mechanisms by which it can interfere with mitochondria to activate ROS production.

Several cellular pathways have been demonstrated to be activated by hypoxia reoxygenation (12, 21). A relationship between ceramide level and ischemia or ischemia reperfusion has been shown in different models. During acute renal failure in mice, ischemia causes decreases of ceramide, whereas during reperfusion, ceramide increases (55). In the hippocampus of gerbils, transient forebrain ischemia, induced an increase in ceramide level (32). An ischemic insult accelerates the degradation of gangliosides and causes an accumulation of ceramide in the ischemic human brain (28). The relationship between increased ceramide production and apoptosis in the intact heart submitted to ischemia/reperfusion has also been demonstrated (4). Ceramide has also been shown as a second messenger of hypoxia/reoxygenation in different cell types, even if different ceramide-related mechanisms have been proposed for explaining their role in hypoxia/reperfusion-induced cellular changes in kidney (55), in brain (32), in HUVEC (58), or in cardiac myocytes (22, 46). Early activation of a sphingomyelinase (SMase) that cleaves membrane sphingomyelin (SM) resulting in the formation of ceramide has been found by Schutze and coworkers (45), who demonstrated that in Jukak T lymphocytes, binding of TNF-{alpha} to its receptor is followed by a rapid activation of an ASMase, with subsequent SM hydrolysis and ceramide production within 3 min. In HUVEC exposed to TNF-{alpha}, ROS production has also been shown to be ceramide dependent (9).

Several results in the present study demonstrated the important role of the ceramide pathway in H/R-induced ROS production. First, we inhibited ROS production by desipramine, an inhibitor of SMase, which is responsible for ceramide production from SM. We also confirmed by direct dosage that H/R induced an increase in ceramide. Interestingly, this increase began in the hypoxic phase of the experiment. This is in line with the studies previously cited, showing that hypoxia can be responsible for the increase in ceramide. Note, however, that no ROS formation has been found during this period of the experiment. Further studies will be necessary to know whether this implies that either ROS formation is largely delayed from ceramide formation or whether it required the oxygen, which is provided during the reoxygenation period. We also confirmed the role of ceramides by demonstrating that exposure of HUVEC to ceramide (N-acylsphingosine) induced a ROS production. This result is similar to that found by others in neuronally differentiated PC12 cells (40) or in human myeloid leukemia U937 cells (13). In addition, we found that the effect of ceramide was inhibited by antimycin A and stigmatellin, which inhibit the mitochondrial respiratory chain complex III, which have been recently found to inhibit H/R-induced ROS production in HUVEC (50). It can be noted that ROS generated at the complex III of the mitochondrial respiratory chain has also been proposed to be an early major mediator in ceramide-induced apoptosis in human myeloid leukemia U937 cells (40).

Different mechanisms by which ceramide can increase ROS production have been proposed. In rat heart mitochondria, ceramide can directly inhibit mitochondrial respiratory chain function and generate ROS by decreasing the activity of the complex III. Gudz et al. (19) hypothesized that ceramide bound to complex III affects cyt b reoxidation in the Q-cycle with subsequent generation of oxygen radicals. In another way, Garcia-Ruiz et al. (14) hypothesized that in rat liver mitochondria, ceramides interact with components of the complex III of the electron transport chain, favoring the production of ROS. Alternatively, in HL60 cells, the effect of ceramide has been shown to involve the activation of a MAPK (41). Such a mechanism has also been reported by Corda et al. (9), showing that TNF-{alpha} induced mitochondrial ROS production in HUVEC occurred via a CAPK. However, in the present experiments DMAP did not inhibit the H/R-induced ROS production, thus making it unlikely that this effect was due to a CAPK.

Other mechanisms by which ceramide can indirectly interfere with mitochondrial ROS production have been proposed. The MPT caused by the opening of a large proteinaceous pore across the inner and outer membrane of mitochondria has been shown to be a causative event in cell injury by a variety of conditions, including ischemia (11, 24, 38). In the L929 line of mouse fibroblasts, MPT occurs as a consequence of the formation of ceramide, which acts to promote induction of the MPT (39). The possibility that H/R-induced mitochondrial ROS production could involve MPT has been explored in the present study. Indeed, no inhibition of H/R-induced ROS production has been found when cyclosporin A, a potent inhibitor of the inner membrane permeability transition, has been used (5). This result is in line with the observation made by Garcia-Ruiz and coworkers (14) who found that CsA did not affect the ROS formation in the presence of ceramide and concluded that the induction of H2O2 induced by ceramide is not the consequence of increased membrane permeability. The present results are also in line with those of several authors suggesting that MPT might be a consequence rather than a cause of ROS production (20, 37, 54, 56).

In the present study, we showed that the antiapoptotic protein Bcl-2 inhibited the H/R-induced ROS production. It should be stressed that a limitation of the present study is that we used external Bcl-2 rather than cells overexpressing Bcl-2. However, our observation is in line with several previous studies. Indeed, different authors have suggested a Bcl-2-mediated reduction of ROS production (26, 56). It has been proposed that its apparent antioxidant function (23, 26) was not due to a direct electron-scavenging or superoxide-metabolizing activity of Bcl-2 itself but rather to the prevention of O2°– production (6). Indeed, the effect of Bcl-2 in H/R-induced ROS production observed in the present study might be explained when considering the following: 1) ceramides can induce cyt c release, as shown in cell cultures (7, 49, 56) and isolated mitochondria (2, 10, 17). 2) Release of cyt c from mitochondria produces a gap in the hierarchically arranged mitochondrial respiratory complex III and IV, and therefore a site for electron leakage (17). In line with the study by Cai and Jones (6), Ghafourifar et al. (17) hypothesized that superoxide formation by mitochondria is a consequence, and not the cause, of cyt c loss. 3) Cyt c release was preventable by preincubation with or overexpression of the antideath protein, Bcl-2 (15, 17, 57).

In conclusion, the present study demonstrated that in endothelial cells submitted to hypoxia and glucose depletion followed by reoxygenation with glucose, the pathway implicated in mitochondrial complex III ROS production is ceramide-dependent and inhibited by the antiapoptotic protein Bcl-2.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. Vicaut, Laboratoire d'Etude de la Microcirculation, Université Paris 7 10, avenue de Verdun, 75010 Paris, France (e-mail: eric.vicaut{at}lrb.aphp.fr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Armstrong JS and Jones DP. Glutathione depletion enforces the mitochondrial permeability transition and causes cell death in Bcl-2 overexpressing HL60 cells. FASEB J 16: 1263–1265, 2002.[Abstract/Free Full Text]
  2. Arora AS, Jones BJ, Patel TC, Bronk SF, and Gores GJ. Ceramide induces hepatocyte cell death through disruption of mitochondrial function in the rat. Hepatology 25: 958–963, 1997.[CrossRef][ISI][Medline]
  3. Bielawska A, Perry DK, and Hannun YA. Determination of ceramides and diglycerides by the diglyceride kinase assay. Anal Biochem 298: 141–150, 2001.[CrossRef][ISI][Medline]
  4. Bielawska AE, Shapiro JP, Jiang L, Melkonyan HS, Piot C, Wolfe CL, Tomei LD, Hannun YA, and Umansky SR. Ceramide is involved in triggering of cardiomyocyte apoptosis induced by ischemia and reperfusion. Am J Pathol 151: 1257–1263, 1997.[Abstract]
  5. Broekemeier KM, Dempsey ME, and Pfeiffer DR. Cyclosporin A is a potent inhibitor of the inner membrane permeability transition in liver mitochondria. J Biol Chem 264: 7826–7830, 1989.[Abstract/Free Full Text]
  6. Cai J and Jones DP. Superoxide in apoptosis. Mitochondrial generation triggered by cytochrome c loss. J Biol Chem 273: 11401–11404, 1998.[Abstract/Free Full Text]
  7. Castedo M, Hirsch T, Susin SA, Zamzami N, Marchetti P, Macho A, and Kroemer G. Sequential acquisition of mitochondrial and plasma membrane alterations during early lymphocyte apoptosis. J Immunol 157: 512–521, 1996.[Abstract]
  8. Chen Q, Vazquez EJ, Moghaddas S, Hoppel CL, and Lesnefsky EJ. Production of reactive oxygen species by mitochondria: central role of complex III. J Biol Chem 278: 36027–36031, 2003.[Abstract/Free Full Text]
  9. Corda S, Laplace C, Vicaut E, and Duranteau J. Rapid reactive oxygen species production by mitochondria in endothelial cells exposed to tumor necrosis factor-alpha is mediated by ceramide. Am J Respir Cell Mol Biol 24: 762–768, 2001.[Abstract/Free Full Text]
  10. Di Paola M, Cocco T, and Lorusso M. Ceramide interaction with the respiratory chain of heart mitochondria. Biochemistry 39: 6660–6668, 2000.[CrossRef][Medline]
  11. Duchen MR, McGuinness O, Brown LA, and Crompton M. On the involvement of a cyclosporin A sensitive mitochondrial pore in myocardial reperfusion injury. Cardiovasc Res 27: 1790–1794, 1993.[Free Full Text]
  12. Fernandez-Checa JC, Garcia-Ruiz C, Colell A, Morales A, Mari M, Miranda M, and Ardite E. Oxidative stress: role of mitochondria and protection by glutathione. Biofactors 8: 7–11, 1998.[ISI][Medline]
  13. France-Lanord V, Brugg B, Michel PP, Agid Y, and Ruberg M. Mitochondrial free radical signal in ceramide-dependent apoptosis: a putative mechanism for neuronal death in Parkinson's disease. J Neurochem 69: 1612–1621, 1997.[ISI][Medline]
  14. Garcia-Ruiz C, Colell A, Mari M, Morales A, and Fernandez-Checa JC. Direct effect of ceramide on the mitochondrial electron transport chain leads to generation of reactive oxygen species Role of mitochondrial glutathione. J Biol Chem 272: 11369–11377, 1997.[Abstract/Free Full Text]
  15. Geley S, Hartmann BL, and Kofler R. Ceramides induce a form of apoptosis in human acute lymphoblastic leukemia cells that is inhibited by Bcl-2, but not by CrmA. FEBS Lett 400: 15–18, 1997.[CrossRef][ISI][Medline]
  16. Genova ML, Ventura B, Giuliano G, Bovina C, Formiggini G, Parenti Castelli G, and Lenaz G. The site of production of superoxide radical in mitochondrial Complex I is not a bound ubisemiquinone but presumably iron-sulfur cluster N2. FEBS Lett 505: 364–368, 2001.[CrossRef][ISI][Medline]
  17. Ghafourifar P, Klein SD, Schucht O, Schenk U, Pruschy M, Rocha S, and Richter C. Ceramide induces cytochrome c release from isolated mitochondria. Importance of mitochondrial redox state. J Biol Chem 274: 6080–6084, 1999.[Abstract/Free Full Text]
  18. Granger DN and Korthuis RJ. Physiologic mechanisms of postischemic tissue injury. Annu Rev Physiol 57: 311–332, 1995.[ISI][Medline]
  19. Gudz TI, Tserng KY, and Hoppel CL. Direct inhibition of mitochondrial respiratory chain complex III by cell-permeable ceramide. J Biol Chem 272: 24154–24158, 1997.[Abstract/Free Full Text]
  20. Halestrap AP, Woodfield KY, and Connern CP. Oxidative stress, thiol reagents, and membrane potential modulate the mitochondrial permeability transition by affecting nucleotide binding to the adenine nucleotide translocase. J Biol Chem 272: 3346–3354, 1997.[Abstract/Free Full Text]
  21. Hannun YA. The sphingomyelin cycle and the second messenger function of ceramide. J Biol Chem 269: 3125–3128, 1994.[Free Full Text]
  22. Hernandez OM, Discher DJ, Bishopric NH, and Webster KA. Rapid activation of neutral sphingomyelinase by hypoxia-reoxygenation of cardiac myocytes. Circ Res 86: 198–204, 2000.[Abstract/Free Full Text]
  23. Hockenbery DM, Oltvai ZN, Yin XM, Milliman CL, and Korsmeyer SJ. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75: 241–251, 1993.[CrossRef][ISI][Medline]
  24. Hoek JB, Farber JL, Thomas AP, and Wang X. Calcium ion-dependent signalling and mitochondrial dysfunction: mitochondrial calcium uptake during hormonal stimulation in intact liver cells and its implication for the mitochondrial permeability transition. Biochim Biophys Acta 1271: 93–102, 1995.[Medline]
  25. Iwata S, Lee JW, Okada K, Lee JK, Iwata M, Rasmussen B, Link TA, Ramaswamy S, and Jap BK. Complete structure of the 11-subunit bovine mitochondrial cytochrome bc1 complex. Science 281: 64–71, 1998.[Abstract/Free Full Text]
  26. Kane DJ, Sarafian TA, Anton R, Hahn H, Gralla EB, Valentine JS, Ord T, and Bredesen DE. Bcl-2 inhibition of neural death: decreased generation of reactive oxygen species. Science 262: 1274–1277, 1993.[Abstract/Free Full Text]
  27. Kluck RM, Bossy-Wetzel E, Green DR, and Newmeyer DD. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275: 1132–1136, 1997.[Abstract/Free Full Text]
  28. Kubota M, Kitahara S, Shimasaki H, and Ueta N. Accumulation of ceramide in ischemic human brain of an acute case of cerebral occlusion. Jpn J Exp Med 59: 59–64, 1989.[Medline]
  29. Li Y and Trush MA. Diphenyleneiodonium, an NAD(P)H oxidase inhibitor, also potently inhibits mitochondrial reactive oxygen species production. Biochem Biophys Res Commun 253: 295–299, 1998.[CrossRef][ISI][Medline]
  30. Malagarie-Cazenave S, Segui B, Leveque S, Garcia V, Carpentier S, Altie MF, Brouchet A, Gouaze V, Andrieu-Abadie N, Barreira Y, Benoist H, and Levade T. Role of FAN in tumor necrosis factor-alpha and lipopolysaccharide-induced interleukin-6 secretion and lethality in D-galactosamine-sensitized mice. J Biol Chem 279: 18648–18655, 2004.[Abstract/Free Full Text]
  31. Marino MW, Dunbar JD, Wu LW, Ngaiza JR, Han HM, Guo D, Matsushita M, Nairn AC, Zhang Y, Kolesnick R, Jaffe EA, and Donner DB. Inhibition of tumor necrosis factor signal transduction in endothelial cells by dimethylaminopurine. J Biol Chem 271: 28624–28629, 1996.[Abstract/Free Full Text]
  32. Nakane M, Kubota M, Nakagomi T, Tamura A, Hisaki H, Shimasaki H, and Ueta N. Lethal forebrain ischemia stimulates sphingomyelin hydrolysis and ceramide generation in the gerbil hippocampus. Neurosci Lett 296: 89–92, 2000.[CrossRef][ISI][Medline]
  33. O'Donnell BV, Tew DG, Jones OT, and England PJ. Studies on the inhibitory mechanism of iodonium compounds with special reference to neutrophil NADPH oxidase. Biochem J 290: 41–49, 1993.[ISI][Medline]
  34. Oh KW, Qian T, Brenner DA, and Lemasters JJ. Salicylate enhances necrosis and apoptosis mediated by the mitochondrial permeability transition. Toxicol Sci 73: 44–52, 2003.[Abstract/Free Full Text]
  35. Palmer G, Horgan DJ, Tisdale H, Singer TP, and Beinert H. Studies on the respiratory chain-linked reduced nicotinamide adenine dinucleotide dehydrogenase. XIV Location of the sites of inhibition of rotenone, barbiturates, and piericidin by means of electron paramagnetic resonance spectroscopy. J Biol Chem 243: 844–847, 1968.[Abstract/Free Full Text]
  36. Park JH, Okayama N, Gute D, Krsmanovic A, Battarbee H, and Alexander JS. Hypoxia/aglycemia increases endothelial permeability: role of second messengers and cytoskeleton. Am J Physiol Cell Physiol 277: C1066–C1074, 1999.[Abstract/Free Full Text]
  37. Pastorino JG, Marcineviciute A, Cahill A, and Hoek JB. Potentiation by chronic ethanol treatment of the mitochondrial permeability transition. Biochem Biophys Res Commun 265: 405–409, 1999.[CrossRef][ISI][Medline]
  38. Pastorino JG, Snyder JW, Serroni A, Hoek JB, and Farber JL. Cyclosporin and carnitine prevent the anoxic death of cultured hepatocytes by inhibiting the mitochondrial permeability transition. J Biol Chem 268: 13791–13798, 1993.[Abstract/Free Full Text]
  39. Pastorino JG, Simbula G, Yamamoto K, Glascott PA Jr, Rothman RJ and Farber JL. The cytotoxicity of tumor necrosis factor depends on induction of the mitochondrial permeability transition. J Biol Chem 271: 29792–29798, 1996.[Abstract/Free Full Text]
  40. Quillet-Mary A, Jaffrezou JP, Mansat V, Bordier C, Naval J, and Laurent G. Implication of mitochondrial hydrogen peroxide generation in ceramide-induced apoptosis. J Biol Chem 272: 21388–21395, 1997.[Abstract/Free Full Text]
  41. Raines MA, Kolesnick RN, and Golde DW. Sphingomyelinase and ceramide activate mitogen-activated protein kinase in myeloid HL-60 cells. J Biol Chem 268: 14572–14575, 1993.[Abstract/Free Full Text]
  42. Ruvolo PP, Deng X, Ito T, Carr BK, and May WS. Ceramide induces Bcl2 dephosphorylation via a mechanism involving mitochondrial PP2A. J Biol Chem 274: 20296–20300, 1999.[Abstract/Free Full Text]
  43. Sanchez-Alcazar JA, Schneider E, Martinez MA, Carmona P, Hernandez-Munoz I, Siles E, De La Torre P, Ruiz-Cabello J, Garcia I, and Solis-Herruzo J.A. Tumor necrosis factor-alpha increases the steady-state reduction of cytochrome b of the mitochondrial respiratory chain in metabolically inhibited L929 cells. J Biol Chem 275: 13353–13361, 2000.[Abstract/Free Full Text]
  44. Sawada GA, Raub TJ, Decker DE, and Buxser SE. Analytical and numerical techniques for the evaluation of free radical damage in cultured cells using scanning laser microscopy. Cytometry 25: 254–262, 1996.[CrossRef][ISI][Medline]
  45. Schutze S, Potthoff K, Machleidt T, Berkovic D, Wiegmann K, and Kronke M. TNF activates NF-kappa B by phosphatidylcholine-specific phospholipase C-induced "acidic" sphingomyelin breakdown. Cell 71: 765–776, 1992.[CrossRef][ISI][Medline]
  46. Semenza GL. Cellular and molecular dissection of reperfusion injury: ROS within and without. Circ Res 86: 117–118, 2000.[Free Full Text]
  47. Shiemke AK, Arp DJ, and Sayavedra-Soto LA. Inhibition of membrane-bound methane monooxygenase and ammonia monooxygenase by diphenyliodonium: implications for electron transfer. J Bacteriol 186: 928–937, 2004.[Abstract/Free Full Text]
  48. Sinaasappel M, van Iterson M, and Ince C. Microvascular oxygen pressure in the pig intestine during haemorrhagic shock and resuscitation. J Physiol 514: 245–253, 1999.[Abstract/Free Full Text]
  49. Susin SA, Zamzami N, Castedo M, Daugas E, Wang HG, Geley S, Fassy F, Reed JC, and Kroemer G. The central executioner of apoptosis: multiple connections between protease activation and mitochondria in Fas/APO-1/CD95- and ceramide-induced apoptosis. J Exp Med 186: 25–37, 1997.[Abstract/Free Full Text]
  50. Therade-Matharan S, Laemmel E, Duranteau J, and Vicaut E. Reoxygenation after hypoxia and glucose depletion causes reactive oxygen species production by mitochondria in HUVEC. Am J Physiol Regul Integr Comp Physiol 287: R1037–R1043, 2004.[Abstract/Free Full Text]
  51. Vanden Hoek TL, Li C, Shao Z, Schumacker PT, and Becker LB. Significant levels of oxidants are generated by isolated cardiomyocytes during ischemia prior to reperfusion. J Mol Cell Cardiol 29: 2571–2583, 1997.[CrossRef][ISI][Medline]
  52. Xu J, Yeh CH, Chen S, He L, Sensi SL, Canzoniero LM, Choi DW, and Hsu CY. Involvement of de novo ceramide biosynthesis in tumor necrosis factor-alpha/cycloheximide-induced cerebral endothelial cell death. J Biol Chem 273: 16521–16526, 1998.[Abstract/Free Full Text]
  53. Yang J, Liu X, Bhalla K, Kim CN, Ibrado AM, Cai J, Peng TI, Jones DP, and Wang X. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275: 1129–1132, 1997.[Abstract/Free Full Text]
  54. Yang JC and Cortopassi GA. Induction of the mitochondrial permeability transition causes release of the apoptogenic factor cytochrome c. Free Radic Biol Med 24: 624–631, 1998.[CrossRef][ISI][Medline]
  55. Zager RA, Iwata M, Conrad DS, Burkhart KM, and Igarashi Y. Altered ceramide and sphingosine expression during the induction phase of ischemic acute renal failure. Kidney Int 52: 60–70, 1997.[ISI][Medline]
  56. Zamzami N, Marchetti P, Castedo M, Decaudin D, Macho A, Hirsch T, Susin SA, Petit PX, Mignotte B, and Kroemer G. Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death. J Exp Med 182: 367–377, 1995.[Abstract/Free Full Text]
  57. Zhang J, Alter N, Reed JC, Borner C, Obeid LM, and Hannun YA. Bcl-2 interrupts the ceramide-mediated pathway of cell death. Proc Natl Acad Sci USA 93: 5325–5328, 1996.[Abstract/Free Full Text]
  58. Zhao H, Miller M, Pfeiffer K, Buras JA, and Stahl GL. Anoxia and reoxygenation of human endothelial cells decrease ceramide glucosyltransferase expression and activates caspases. FASEB J 17: 723–724, 2003.[Abstract/Free Full Text]
  59. Zweier JL. Measurement of superoxide-derived free radicals in the reperfused heart. Evidence for a free radical mechanism of reperfusion injury. J Biol Chem 263: 1353–1357, 1988.[Abstract/Free Full Text]
  60. Zweier JL, Kuppusamy P, Thompson-Gorman S, Klunk D, and Lutty GA. Measurement and characterization of free radical generation in reoxygenated human endothelial cells. Am J Physiol Cell Physiol 266: C700–C708, 1994.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J Am Coll CardiolHome page
A. Jabs, S. Gobel, P. Wenzel, A. L. Kleschyov, M. Hortmann, M. Oelze, A. Daiber, and T. Munzel
Sirolimus-induced vascular dysfunction increased mitochondrial and nicotinamide adenosine dinucleotide phosphate oxidase-dependent superoxide production and decreased vascular nitric oxide formation.
J. Am. Coll. Cardiol., June 3, 2008; 51(22): 2130 - 2138.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
N. Baudry, E. Laemmel, and E. Vicaut
In vivo reactive oxygen species production induced by ischemia in muscle arterioles of mice: involvement of xanthine oxidase and mitochondria
Am J Physiol Heart Circ Physiol, February 1, 2008; 294(2): H821 - H828.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
D. X. Zhang and D. D. Gutterman
Mitochondrial reactive oxygen species-mediated signaling in endothelial cells
Am J Physiol Heart Circ Physiol, May 1, 2007; 292(5): H2023 - H2031.
[Abstract] [Full Text] [PDF]

Home page
 Exp. Biol. Med.Home page
Q. Cheng, T. Nguyen, H. Song, and J. Bonanno
Hypoxia Protects Human Corneal Endothelium from Tertiary Butyl Hydroperoxide and Paraquat-Induced Cell Death In Vitro
Experimental Biology and Medicine, March 1, 2007; 232(3): 445 - 453.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
D. L. Hoffman, J. D. Salter, and P. S. Brookes
Response of mitochondrial reactive oxygen species generation to steady-state oxygen tension: implications for hypoxic cell signaling
Am J Physiol Heart Circ Physiol, January 1, 2007; 292(1): H101 - H108.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (8)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Therade-Matharan, S.
Right arrow Articles by Vicaut, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Therade-Matharan, S.
Right arrow Articles by Vicaut, E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.