Taurine inhibits apoptosis by preventing formation of the Apaf-1/caspase-9 apoptosome

Tomoka Takatani,1 Kyoko Takahashi,1 Yoriko Uozumi,1 Eriko Shikata,1 Yasuhiro Yamamoto,1 Takashi Ito,1 Takahisa Matsuda,1 Stephen W. Schaffer,2 Yasushi Fujio,1 and Junichi Azuma1

1Department of Clinical Evaluation of Medicines and Therapeutics, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan; and 2Department of Pharmacology, University of South Alabama School of Medicine, Mobile, Alabama 36688

Submitted 22 January 2004 ; accepted in final form 3 June 2004


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Cardiomyocyte apoptosis contributes to cell death during myocardial infarction. One of the factors that regulate the degree of apoptosis during ischemia is the amino acid taurine. To study the mechanism underlying the beneficial effect of taurine, we examined the interaction between taurine and mitochondria-mediated apoptosis using a simulated ischemia model with cultured rat neonatal cardiomyocytes sealed in closed flasks. Exposure to medium containing 20 mM taurine reduced the degree of apoptosis following periods of ischemia varying from 24 to 72 h. In the untreated group, simulated ischemia for 24 h led to mitochondrial depolarization accompanied by cytochrome c release. The apoptotic cascade was also activated, as evidenced by the activation of caspase-9 and -3. Taurine treatment had no effect on mitochondrial membrane potential and cytochrome c release; however, it inhibited ischemia-induced cleavage of caspase-9 and -3. Taurine loading also suppressed the formation of the Apaf-1/caspase-9 apoptosome and the interaction of caspase-9 with Apaf-1. These findings demonstrate that taurine effectively prevents myocardial ischemia-induced apoptosis by inhibiting the assembly of the Apaf-1/caspase-9 apoptosome.

ischemia; cultured cardiomyocytes


MYOCARDIAL ISCHEMIA causes depressed myocardial function and associated deleterious morphological alterations that lead to heart failure. This injury is a pathological process that results in extensive cell death, a significant portion of which can be attributed to apoptosis (10). Myocyte apoptosis has been demonstrated in clinical cases of myocardial infarction as well as in rabbit, rat, and mouse models of continuous ischemia or ischemic reperfusion (7, 8, 18). It has been proposed that mitochondrial dysfunction is the primary cause of apoptosis in these animal models of ischemic myopathy (4, 14, 15, 26). A key step in the initiation of apoptosis by the mitochondrial pathway is the release of cytochrome c from the intermembrane space of the mitochondria into the cytoplasm. Because ischemia results in the disruption of the mitochondrial membrane potential ({Delta}{Psi}), the release of cytochrome c is likely triggered by the opening of the mitochondrial permeability transition pore (6, 11, 24). When cytochrome c enters the cytosol, it associates with the apoptotic protease activity factor-1 (Apaf-1) to form a large complex referred to as an apoptosome. The initiator caspase, caspase-9, is activated through a protein-protein interaction with the apoptosome (12). Effector caspases, such as caspase-3, are activated through cleavage by the initiator caspase.

The mitochondrial pathway of apoptosis is very tightly regulated. Although most cytoprotective agents prevent cytochrome c release, recent studies have revealed that other steps in the process are subject to regulation. In the present study, we have examined the mechanism underlying the cardioprotective activity of the {beta}-amino acid, taurine. Taurine (2-aminoethanesulfonic acid) is a ubiquitous substance involved in osmoregulation, modulation of calcium transport, and regulation of oxidative stress, in particular through its ability to scavenge hypochlorous acid (16, 19). Moreover, taurine has been found to prevent high-glucose-mediated endothelial cell apoptosis through its antioxidant property and regulation of intracellular calcium homeostasis (27). Because osmotic stress, calcium overload, and oxidative stress adversely impact mitochondrial function (25), there is reason to suspect that taurine might benefit the cardiomyocyte through the mitochondrial-linked pathway.

In the present study, the interaction between taurine and mitochondria-mediated apoptosis is investigated in a newly developed simulated ischemia model utilizing isolated cardiomyocytes, which are incubated with medium containing and lacking taurine and then sealed within cultured flasks (21). Our findings show that taurine suppresses simulated ischemia-induced apoptosis in cardiomyocytes by targeting the Apaf-1/caspase-9 apoptosome.


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Cell cultures and the newly simulated ischemia model. Primary cardiomyocyte cultures from 1-day-old Wistar rats were prepared according to the procedure described previously (21). All experimental procedures were approved by the Animal Care Committee of Osaka University and conformed to international guidelines. The myocytes were plated onto 12.5-cm2 flasks (Falcon) at a density of confluence in culture medium, which consisted of a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 (containing 3,151 mg/l dextrose; ICN Biomedicals) supplemented with 5% newborn calf serum (ICN Biomedicals), 3 mM pyruvic acid, 100 µM ascorbic acid, 5 µg/ml insulin, 5 µg/ml transferrin, and 5 ng/ml selenium (Boehringer Mannheim). This procedure yielded cell preparations containing 90–95% myocytes, as assessed by microscopic observation of cell beating (data not shown). These cells were maintained for 2 days with medium changed daily and were serum-starved for 24 h before the experiments. For seal-induced cardiac ischemia, the flask medium was first replaced with serum-free culture medium (2.5 ml, containing 3,151 mg/l dextrose) and filled with phosphate-buffered saline (PBS; 47.5 ml) containing the following constituents (in mM): 137 NaCl, 8.1 Na2HPO4·12 H2O, 2.7 KCl, 1.5 KH2PO4, 0.9 CaCl2·2H2O, and 0.3 MgCl2·12H2O. Next, the flasks were bubbled with 5% CO2-95% N2 for 2 min to fix the initial pH at 7.4 and to eliminate oxygen from the remaining air space. Subsequently, the lid was tightly sealed to prevent gas from entering the flask, and flasks were incubated at 37°C. For controls, the cells were replaced with 2.5 ml of serum-containing culture medium and incubated at 37°C in 95% air-5% CO2. The culture medium in the control group was changed daily. The simulated ischemia model mimics the clinical stresses of ischemia, including the stresses of hypoxia, acidosis, and stagnant incubation medium.

Detection of apoptotic cells. To visualize fragmented nuclei, we fixed cells with 1% paraformaldehyde for 30 min at room temperature. After being rinsed in PBS, the cells were permeabilized in 70% ethanol. The cells were rinsed twice in PBS and stained with a fluorescent dye, Hoechst 33258 (Sigma-Aldrich), for 15 min at room temperature. After a final rinse in PBS, the cells were mounted in the FlowFade antifade reagent (Molecular Probes) and visualized under ultraviolet light with the Olympus fluorescence microscopy system. More than 100 cardiomyocytes obtained from 3 different primary culture preparations were counted. The percentage of apoptotic nuclei was calculated as the ratio of fragmented nuclei to the total amount of nuclei. Further evaluation of apoptosis was performed with a commercially available cell death detection kit to find DNA strand breaks using the terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling (TUNEL) reagent according to the manufacturer's protocol (Promega). Cells that showed positive TUNEL staining in the nuclei were identified as apoptotic.

Measurement of mitochondrial membrane potential. Loss of {Delta}{Psi} was assessed using a fluorescent dye, the lipophilic cationic probe JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine; Intergen). Cells were incubated with 5 µg/ml JC-1 for 15 min at 37°C and examined with the Olympus fluorescence microscopy system. The excitation wavelength was 488 nm, and the emission fluorescence for JC-1 was monitored at 530 and 590 nm. The red emission of the dye is attributable to a potential-dependent aggregation in the mitochondria, reflecting {Delta}{Psi}. Green fluorescence reflects the monomeric form of JC-1, appearing in the cytosol after mitochondrial membrane depolarization.

Preparation of mitochondrial and cytosolic fractions and total cell lysates. Mitochondrial and cytosolic fractions were prepared from nontreated or taurine-treated cells cultured under nonischemic or ischemic conditions. Preparation of mitochondrial and cytosolic fractions was achieved using a commercially available mitochondria/cytosol fractionation kit according to the manufacturer's protocol (BioVision). The cytosolic and mitochondrial fractions were stored at –80°C. Total cellular proteins were extracted in lysis buffer containing 150 mM NaCl, 10 mM Tris (pH 7.4), and 1 mM EDTA, plus 1% Triton X-100, 1% deoxycholic acid and protease inhibitor mixture (Sigma-Aldrich), followed by centrifugation at 1,500 g for 20 min. Protein concentrations were determined by the method of Lowry et al. (13), using bovine serum albumin as a standard.

Western blot analysis. Proteins (12 µg) from the mitochondrial fraction, cytosolic fraction, or total cell lysate were analyzed by SDS-PAGE (12.5 or 14% gel). After blotting, the Immobilon-P membrane (Millipore) was blocked with 5% BSA in Tween 20 in PBS at room temperature for 1 h. Immunoblots were incubated at room temperature for 60 min with the specific primary antibody to cytochrome c (PharMingen) or Apaf-1 (BioVision). After further washing, the membranes were incubated for 1 h with the secondary antibody (horseradish peroxidase conjugated). The enhanced chemiluminescence procedure was used for detection of the bands (ECL; Santa Cruz Biotechnology). Blots were reprobed with antibody to actin (Sigma-Aldrich) as a loading control. Quantitative analysis of immunoblotted bands was performed by computer program (NIH Image, version 1.61).

Detection of caspase-3 and -9 immunoreactivity. Total cell lysate (12 µg) was separated by 14% SDS-PAGE and then subjected to Western blot analysis with the use of antibody against caspase-3 or -9 (Santa Cruz Biotechnology).

Immunoprecipitation. For determining the Apaf-1-caspase-9 interaction, the cell lysates were prepared in lysis buffer containing 150 mM NaCl, 10 mM Tris (pH 7.4), and 1 mM EDTA, plus 1% Triton X-100, 1% deoxycholic acid, and protease inhibitor mixture. After homogenization and centrifugation, the supernatants were immunoprecipitated with antibody against Apaf-1 (1:150 dilution; Santa Cruz Biotechnology) plus 10 µl of protein A-Sepharose (Santa Cruz Biotechnology) for 5 h. Immunoprecipitates were washed, separated by 12.5% SDS-PAGE, and then subjected to Western blot analysis with the use of antibody against either Apaf-1 or caspase-9 (Medical & Biological Laboratories).

Determination of ATP content. The ATP content of the myocytes was measured according to the manufacturer's instructions, using a commercially available ATP assay kit (Toyo) The results are expressed in picomoles per microgram of protein. Protein concentration was determined by the method of Lowry et al. (13), using bovine serum albumin as a standard.

Statistical analysis. Depending on the design of the experiment, statistical significance was determined using the Student's t-test, {chi}2 test, or analysis of variance, with the Bonferroni method being used to compare individual data points for a significant F value. Each value was expressed as a mean ± SE. Differences were considered significant when the calculated P value was <0.05.


    RESULTS
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Taurine suppresses ischemia-induced apoptosis in cultured neonatal rat cardiomyocytes. In agreement with a previous finding from our laboratory (22), we found that isolated neonatal cardiomyocytes showed resistance to ischemia-induced apoptosis when exposed to medium containing 20 mM taurine. Figure 1A shows a quantitative determination of apoptotic nuclei in each experimental group. Cells incubated for the 24- to 72-h ischemic period were stained by Hoechst 33258, and the apoptotic nuclei were identified by the characteristic condensed, fragmented nuclei. Exposure of the cells to medium containing 20 mM taurine reduced the frequency of apoptosis after 24, 48, and 72 h of ischemia from 16 to 6%, from 26 to 13%, and from 42 to 15%, respectively. Figure 1B shows representative photomicrographs of results of TUNEL assay and Hoechst 33258 staining after a 72-h ischemic insult. Similar results were shown by TUNEL indicating that taurine significantly decreased apoptosis by ~35% (from 29 to 10%, P < 0.01) after a 72-h ischemic insult.



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Fig. 1. Taurine suppresses ischemia-induced apoptosis in cultured neonatal rat cardiomyocytes. A: percentage of cells undergoing apoptosis as measured by Hoechst 33258 stain. Samples (n = 360–949 cells) were obtained from 3 different primary culture preparations. #P < 0.01 vs. control group. *P < 0.05, **P < 0.01 vs. ischemia group at 24–72 h. B: photomicrographs show TUNEL staining (a–c; magnification x800) and Hoechst 33258 staining (d–f; magnification x600). Cells in a and d were exposed to normoxia for 72 h, cells in b and e were exposed to ischemia for 72 h in the absence of taurine, and cells in c and f were exposed to ischemia for 72 h in the presence of 20 mM taurine.

 
Influence of taurine on ischemia-induced mitochondrial dysfunction. One of the mechanisms contributing to mitochondrial-mediated apoptosis is the activation of the mitochondrial permeability transition pore, an event associated with a decrease in membrane potential ({Delta}{Psi}) and the loss of cytochrome c from the mitochondria. To determine whether taurine affected the permeability pore, we assessed {Delta}{Psi} in ischemic cardiomyocytes using the potential-sensitive fluorescent probe JC-1. Whereas control cells exhibited punctate red staining (Fig. 2A, top) indicative of coupled mitochondria with a normal {Delta}{Psi}, ischemic myocytes developed a diffuse green staining pattern, representative of reduced {Delta}{Psi}. Although taurine treatment was cardioprotective, it had no effect on JC-1 staining (Fig. 2A, middle and bottom). Moreover, cells placed in medium containing 20 mM taurine showed no change in the levels of cytochrome c released into the cytosol. Figure 2B reveals that cytochrome c was primarily localized to the mitochondria of cardiomyocytes incubated under normoxic conditions. However, the mitochondrial cytochrome c content decreased ~75% in cardiomyocytes placed for 24 h in sealed containers, with the degree of cytochrome c release being the same for cells treated with or without taurine (Fig. 2C).



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Fig. 2. Influence of taurine on ischemia-induced mitochondrial dysfunction of cultured cardiomyocytes. A: mitochondrial membrane potential ({Delta}{Psi}) of cardiomyocytes exposed to ischemia for 24 h in the absence (–) or presence (+) of 20 mM taurine was determined using the potential-sensitive fluorescent probe JC-1. Each panel shows an overlay of 2 images; orange-yellow color denotes colocalization of red (aggregate) and green (monomer) fluorescence signals. Results are representative of 1 experiment from a total of 3 experiments performed. B: cardiomyocytes were exposed to ischemia for 24 h in the absence or presence of 20 mM taurine. Proteins from mitochondrial and cytosolic fractions were immunoblotted with anti-cytochrome c antibody. C: quantitative analysis of cytochrome c release from the mitochondria into the cytosol. This experiment was repeated twice and similar results were obtained. Data represent the results from 1 experiment.

 
Taurine inhibits the activation of caspase-9 and -3 in ischemic cardiomyocytes. To examine the effects of taurine on the apoptotic machinery located downstream from the mitochondria, we initially examined the activation states of caspase-9 and -3 using Western blot analysis. No apparent activation of the two caspases was observed after a 24-h ischemic insult. However, after 30 h of ischemia, the active cleavage product of caspase-9 was detected in the untreated cells but not in cells treated with 20 mM taurine (Fig. 3A). Figure 3B shows that ischemia-mediated activation of caspase-3, the effector caspase located downstream from caspase-9, is also inhibited by inclusion of 20 mM taurine in the incubation medium during the ischemic insult.



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Fig. 3. Taurine prevents ischemia-induced caspase-9 and -3 processing in cultured cardiomyocytes. Cardiomyocytes were exposed to ischemia for 30 h in the absence or presence of 20 mM taurine. Top: active subunits p35 and p17 of caspase-9 and caspase-3, respectively, were assessed by immunoblot analysis using antibodies directed against either caspase-9 (A) or caspase-3 (B). Bottom: membranes were stripped and reprobed with antibody against actin. Representative results of 3 independent experiments are shown.

 
Regulation of the Apaf-1 apoptosome by taurine. The prevention of ischemia-mediated activation of caspase-9 and -3 without alterations in cytochrome c release implies that taurine must inhibit the activation of caspase-9 by the Apaf-1/caspase-9 apoptosome. To evaluate this hypothesis, we examined the formation of the Apaf-1/caspase-9 complex by monitoring the coprecipitation of Apaf-1 and caspase-9 using the Apaf-1 antibody. Figure 4 shows the effect of taurine treatment on the formation of the Apaf-1/caspase-9 complex in cardiomyocytes after a 24-h ischemic insult. In the untreated cells, simulated ischemia facilitated the formation of the oligomeric Apaf-1/caspase-9 apoptosome, as evidenced by the 60% increase in the amount of caspase-9 that coprecipitated with Apaf-1 (Fig. 4, left, and Fig. 5A). However, taurine treatment markedly reduced the amount of caspase-9 associated with Apaf-1 during ischemia. On the basis of Western blot analysis of Apaf-1, the effect of taurine could not be attributed to a decrease in the expression of Apaf-1 (Fig. 4, right, and Fig. 5B). Moreover, taurine treatment had no effect on intracellular ATP content, a factor also contributing to the assembly of the apoptosome (Fig. 5C).



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Fig. 4. Taurine inhibits formation of Apaf-1/caspase-9 complex in cultured cardiomyocytes. Cardiomyocytes were exposed to ischemia for 24 h in the absence or presence of 20 mM taurine. An equal volume of cell lysate was immunoprecipitated (IP) with antibody against Apaf-1, and the precipitate, as well as the cell lysate, was analyzed by immunoblot with antibody against either Apaf-1 or caspase-9. Membranes at bottom right were stripped and reprobed with an antibody directed against actin. Results shown are representative of 3 independent experiments.

 


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Fig. 5. Effect of taurine on Apaf-1/caspase-9 apoptosome formation and intracellular ATP content. A: extent of caspase-9 binding to Apaf-1 after a 24-h ischemic insult. Cardiomyocytes were exposed to ischemia for 24 h in the absence or presence of 20 mM taurine. Cell lysates were immunoprecipitated with antibody against Apaf-1, and the precipitates were analyzed by immunoblot with antibody against either Apaf-1 or caspase-9. Apaf-1 and caspase-9 contents were quantified by computer software. **P < 0.01 vs. control group. *P < 0.05 vs. ischemia group (n = 3–6). B: Apaf-1 expression in cultured cardiomyocytes exposed to simulated ischemia for 24 h in the absence or presence of 20 mM taurine. C: intracellular ATP content in cultured cardiomyocytes exposed to simulated ischemia for 24 h in the absence or presence of 20 mM taurine.

 

    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In the present study, we demonstrated that taurine prevented the ischemia-induced apoptosis in cardiomyocytes, accompanied by the inactivation of caspase-9 and -3. Taurine treatment inhibited Apaf-1/caspase-9 apoptosome formation without preventing mitochondrial dysfunction under ischemic conditions. Thus we proposed that taurine mediates cardiomyocyte protection by regulating Apaf-1/caspase-9 apoptosome formation.

Despite the accumulating data concerning Apaf-1/caspase 9 apoptosome, it remains to be elucidated how taurine inhibits apoptosome formation. There was no difference in the level of intracellular ATP content, cytochrome c release, and Apaf-1 expression between taurine-treated and untreated myocytes after a 24-h ischemic insult. Recent reports (3, 5, 17) have documented that a heat shock protein, HSP70, interacts with Apaf-1 and blocks the assembly of functional apoptosome. To elucidate the involvement of HSP70 in taurine-mediated cytoprotection, the expression of HSP was analyzed by immunoblotting; however, HSP70 was not upregulated by taurine (data not shown). Recently, we have demonstrated that taurine prevents the ischemia-induced apoptosis in cardiomyocytes through Akt (23). Thus it is possible that taurine-mediated activation of Akt negatively regulates Apaf-1/caspase-9 interaction, although further studies are required.

Numerous studies (9, 12, 20, 28) have suggested that Apaf-1 plays a crucial role in mitochondria-mediated apoptosis. Mitochondria-mediated apoptosis is involved in the onset of cardiovascular diseases (4, 14, 15, 26), especially in ischemic heart disease. Taking these findings together with previous reports that the administration of taurine shows efficacy in the treatment of patients with congestive heart failure (1, 2), it could be proposed that analyses of cytoprotective mechanisms of taurine provide novel strategies for the treatment of ischemic heart disease.

In conclusion, our findings show that taurine suppresses ischemia-induced apoptosis in cardiomyocytes by preventing formation of the Apaf-1/caspase-9 apoptosome. This is the first demonstration of the molecular mechanisms for the antiapoptotic effects of taurine.


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This work was partially supported by a grant from Taisho Pharmaceutical Co. Ltd.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. Azuma, Dept. of Clinical Evaluation of Medicines and Therapeutics, Graduate School of Pharmaceutical Sciences, Osaka Univ., Suita, Osaka 565-0871, Japan (E-mail: azuma{at}phs.osaka-u.ac.jp)

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
 METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
1. Azuma J, Sawamura A, and Awata N. Usefulness of taurine in chronic congestive heart failure and its prospective application. Jpn Circ J 56: 95–99, 1992.[ISI][Medline]

2. Azuma J, Sawamura A, Awata N, Ohta H, Hamaguchi T, Harada H, Takihara K, Hasegawa H, Yamagami T, Ishiyama T, Iwata H, and Kishimoto S. Therapeutic effect of taurine in congestive heart failure: a double-blind crossover trial. Clin Cardiol 8: 276–282, 1985.[ISI][Medline]

3. Beere HM, Wolf BB, Cain K, Mosser DD, Mahboubi A, Kuwana T, Tailor P, Morimoto RI, Cohen GM, and Green DR. Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome. Nat Cell Biol 2: 469–475, 2000.[CrossRef][ISI][Medline]

4. Bialik S, Cryns VL, Drincic A, Miyata S, Wollowick AL, Srinivasan A, and Kitsis RN. The mitochondrial apoptotic pathway is activated by serum and glucose deprivation in cardiac myocytes. Circ Res 85: 403–414, 1999.[Abstract/Free Full Text]

5. Cain K, Langlais C, Sun XM, Brown DG, and Cohen GM. Physiological concentrations of K+ inhibit cytochrome c-dependent formation of the apoptosome. J Biol Chem 276: 41985–41990, 2001.[Abstract/Free Full Text]

6. Crompton M. The mitochondrial permeability transition pore and its role in cell death. Biochem J 341: 233–249, 1999.[CrossRef][ISI][Medline]

7. Fliss H and Gattinger D. Apoptosis in ischemic and reperfused rat myocardium. Circ Res 79: 949–956, 1996.[Abstract/Free Full Text]

8. Gottlieb RA, Burleson KO, Kloner RA, Babior BM, and Engler RL. Reperfusion injury induces apoptosis in rabbit cardiomyocytes. J Clin Invest 94: 1621–1628, 1994.[ISI][Medline]

9. Green DR and Reed JC. Mitochondria and apoptosis. Science 281: 1309–1312, 1998.[Abstract/Free Full Text]

10. Kang PM and Izumo S. Apoptosis and heart failure: a critical review of the literature. Circ Res 86: 1107–1113, 2000.[Free Full Text]

11. Kroemer G, Dallaporta B, and Resche-Rigon M. The mitochondrial death/life regulator in apoptosis and necrosis. Annu Rev Physiol 60: 619–642, 1998.[CrossRef][ISI][Medline]

12. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, and Wang X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91: 479–489, 1997.[ISI][Medline]

13. Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–275, 1951.[Free Full Text]

14. Malhotra R and Brosius FC III. Glucose uptake and glycolysis reduce hypoxia-induced apoptosis in cultured neonatal rat cardiac myocytes. J Biol Chem 274: 12567–12575, 1999.[Abstract/Free Full Text]

15. Narula J, Pandey P, Arbustini E, Haider N, Narula N, Kolodgie FD, Dal Bello B, Semigran MJ, Bielsa-Masdeu A, Dec GW, Israels S, Ballester M, Virmani R, Saxena S, and Kharbanda S. Apoptosis in heart failure: release of cytochrome c from mitochondria and activation of caspase-3 in human cardiomyopathy. Proc Natl Acad Sci USA 96: 8144–8149, 1999.[Abstract/Free Full Text]

16. Rasmusson RL, Davis DG, and Lieberman M. Amino acid loss during volume regulatory decrease in cultured chick heart cells. Am J Physiol Cell Physiol 264: C136–C145, 1993.[Abstract/Free Full Text]

17. Saleh A, Srinivasula SM, Balkir L, Robbins PD, and Alnemri ES. Negative regulation of the Apaf-1 apoptosome by Hsp70. Nat Cell Biol 2: 476–483, 2000.[CrossRef][ISI][Medline]

18. Saraste A, Pulkki K, Kallajoki M, Henriksen K, Parvinen M, and Voipio-Pulkki LM. Apoptosis in human acute myocardial infarction. Circulation 95: 320–323, 1997.[Abstract/Free Full Text]

19. Schaffer S, Takahashi K, and Azuma J. Role of osmoregulation in the actions of taurine. Amino Acids 19: 527–546, 2000.[CrossRef][ISI][Medline]

20. Srinivasula SM, Ahmad M, Fernandes-Alnemri T, and Alnemri ES. Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization. Mol Cell 1: 949–957, 1998.[ISI][Medline]

21. Takahashi K, Ohyabu Y, Schaffer SW, and Azuma J. Cellular characterization of an in-vitro cell culture model of seal-induced cardiac ischaemia. J Pharm Pharmacol 53: 379–386, 2001.[ISI][Medline]

22. Takahashi K, Ohyabu Y, Solodushko V, Takatani T, Itoh T, Schaffer SW, and Azuma J. Taurine renders the cell resistant to ischemia-induced injury in cultured neonatal rat cardiomyocytes. J Cardiovasc Pharmacol 41: 726–733, 2003.[CrossRef][ISI][Medline]

23. Takatani T, Takahashi K, Uozumi Y, Matsuda T, Ito T, Schaffer SW, Fujio Y, and Azuma J. Taurine prevents the ischemia-induced apoptosis in cultured neonatal rat cardiomyocytes through Akt/caspase-9 pathway. Biochem Biophys Res Commun 316: 484–489, 2004.[CrossRef][ISI][Medline]

24. Tsujimoto Y. Cell death regulation by the Bcl-2 protein family in the mitochondria. J Cell Physiol 195: 158–167, 2003.[CrossRef][ISI][Medline]

25. Tsujimoto Y and Shimizu S. The voltage-dependent anion channel: an essential player in apoptosis. Biochimie 84: 187–193, 2002.[CrossRef][ISI][Medline]

26. Wang X. The expanding role of mitochondria in apoptosis. Genes Dev 15: 2922–2933, 2001.[Free Full Text]

27. Wu QD, Wang JH, Fennessy F, Redmond HP, and Bouchier-Hayes D. Taurine prevents high-glucose-induced human vascular endothelial cell apoptosis. Am J Physiol Cell Physiol 277: C1229–C1238, 1999.[Abstract/Free Full Text]

28. Zou H, Henzel WJ, Liu X, Lutschg A, and Wang X. Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 90: 405–413, 1997.[ISI][Medline]





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