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Unidad de Investigacion B, Hospital Universitario Vall d'Hebron, Pg. Vall d'Hebron 119–129, 08035 Barcelona, Spain
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(Received 18 May 2004;
accepted after revision 17 June 2004;
first published online 24 June 2004)
Corresponding author D. Garcia-Dorado: Unidad de Investigacion B, Hospital Universitario Vall d'Hebron, Pg. Vall d'Hebron 119–129, 08035 Barcelona, Spain. Email: dgdorado{at}vhebron.net
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Introduction |
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The amino acid glycine (Gly) has been shown to exert a protective effect against cell death after diverse insults including ischaemia–reperfusion injury in different cell types other than cardiomyocytes, but the mechanism of this effect remains elusive (Nishimura et al. 1998; Schemmer et al. 1999; Nishimura & Lemasters, 2001; Dong et al. 2001; Zhang et al. 2003). In this study we investigated the effect of Gly on MPT and reoxygenation-induced cell death in cultured HL-1 cardiac myocytes, intact rat myocardium and rat heart mitochondria. The results show that Gly, an endogenous amino acid, may play a cardioprotective effect in reoxygenated cardiomyocytes by preventing MPT associated with pH normalization.
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Methods |
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Studies in HL-1 cardiomyocytes
HL-1 cardiac myocytes were plated at 20 000 cells cm–2 density in glass-bottomed culture dishes until a 70–80% confluence was achieved, as previously described (Ruiz-Meana et al. 2003).
Simulated ischaemia–reoxygenation. Cell pellets were submitted to 1 h simulated ischaemia (SI) by suspending them in a buffer containing (mM): NaCl 140, KCl 3.6, CaCl2 2, MgSO4 1.2, Hepes 20, NaCN 2, 2-deoxyglucose 20, at pH 6.4, 37°C. Reoxygenation was performed by centrifugation and resuspension of the pellet in a control normoxic buffer at pH 7.4, with 5 mM glucose.
Experimental interventions. The effect of modifying the reoxygenation buffer by lowering its pH (up to 6.4) or by adding Gly (3 mM) was investigated. The role of Ca2+ was studied by sequestration of either intra- or extracellular Ca2+ (addition of 30 µM BAPTA or removal of CaCl2 in the presence of 2 mM EGTA).
Cell volume and viability. Changes in cell volume were determined by analysis of forward scatter (FACscalibur flow cytometer, Becton Dickinson, USA) and cell viability by exclusion of propidium iodide (PI). Double staining with annexinV–fluorescein isothiocyanate (FITC)–PI was used to quantify the contribution of apoptosis (annexin–FITC+–PI– cells) to cell death.
Intracellular pH and Na+. Changes in cytosolic [H+] and [Na+] during SI–reoxygenation were monitored by ratio-fluorescence imaging in HL-1 cells loaded, respectively, with 3 µM BCECF-AM or 5 µM SBFI (Molecular Probes, USA) (Ruiz-Meana et al. 2003).
Studies in isolated rat hearts
Heart preparation. After an intraperitoneal injection of sodium pentobarbital (100 mg kg–1), the hearts from 18 Sprague-Dawley rats (280–350 g) were rapidly removed and perfused with an oxygenated Krebs solution at 37°C, as previously described (Rodriguez-Sinovas et al. 2003).
Experimental protocol. After 40 min of equilibration, hearts were submitted to 2 h of hypoxia at pH 6.4 followed by 1 h of reoxygenation. For hypoxic perfusion, a modified Krebs buffer (mM: NaCl 139.5, KCl 4.7, MgSO4 1.2, CaCl2 2.5, NaHCO3 3.5, KH2PO4 1.2, and sucrose 11) was bubbled with 95% N2–5% CO2. Reoxygenation was performed by switching the hypoxic Krebs solution to a glucose-containing oxygenated (95% O2–5% CO2) Krebs solution. Hearts were allocated to one of three groups: (1) control reoxygenation (n= 6) at pH 7.4; (2) acidic reoxygenation (n= 5) at pH 6.4; (3) reoxygenation in the presence of 10 mM Gly (n= 5). Two additional hearts were used to calculate cumulative concentration–response curves of developed tension and transmembrane action potential to Gly (from 10–4 to 10–1M, each concentration given during 15 min) under normoxia. Hearts were paced from the base (2.5 ms, 4 V pulses at 2.5 Hz) and transmembrane action potentials were recorded from the apex, as described (Rodriguez-Sinovas et al. 2003).
Enzyme release. Lactate dehydrogenase (LDH) release during reoxygenation was measured in samples taken from the effluent as previously described (Rodriguez-Sinovas et al. 2003).
Studies in isolated mitochondria
Isolation of mitochondria. Rat heart mitochondria were isolated by differential centrifugation, according to the method described by Holmuhamedov et al. (1998). Protein was adjusted to 1 mg ml–1.
Experimental interventions. Mitochondria were submitted to either (1) simulated ischaemia (SI)–reoxygenation or (2) 15 min Ca2+ overload. To simulate ischaemia, the mitochondrial pellet was suspended in a metabolic inhibition buffer (mM: KCl 150, NaCl 7, Hepes 6, KH2PO4 0.5, sucrose 50, MgCl2 1, ADP 0.1, CaCl2 0.01 and NaCN 2), at pH 6.4, for 1 h at 37°C. Reoxygenation was performed by switching to a NaCN-free solution containing (mM): KCl 150, NaCl 7, Hepes 6, KH2PO4 0.5, sucrose 50, MgCl2 1, ADP 0.1, CaCl2 0.05, ATP 0.3, succinate 0.3, ascorbic acid 2.5, at pH 7.2. The effects of acid pH (6.4), Gly (3 mM) or cyclosporin A (CsA, 1 µM), as well as substrates (5 mM succinate, 5 mM pyruvate or 5 mML-alanine) on MPT were assessed during the first 30 min of reoxygenation. Also, the contribution of Ca2+ to the MPT was tested by adding 2 mM EGTA or by increasing the extracellular Ca2+ to 200 µM in the reoxygenation buffer. In the control group, mitochondria were submitted to the same experimental manipulations in a NaCN-free, low Ca2+ (0.1 µM) buffer containing (mM): KCl 150, NaCl 7, Hepes 6, KH2PO4 0.5, sucrose 50, MgCl2 1, ADP 0.1, ATP 0.3, succinate 5, ascorbic acid 2.5, at pH 7.2. The inhibitory effect of acid pH and Gly on MPT was also assessed in normoxic mitochondria submitted to 200 µM CaCl2 for 15 min.
Swelling assay. Changes in mitochondrial volume were assayed spectrophotometrically in mitochondrial suspensions at 25°C using either 96-well-plates (Garlid et al. 1996) or 500 µl cuvettes. Light absorbance was determined at 520 nm under basal conditions, after 60 min of SI, and at the end of reoxygenation. Changes in light absorbance were normalized with respect to basal values. In normoxic Ca2+-stressed mitochondria light absorbance was monitored throughout time following addition of 200 µM Ca2+ to the mitochondrial suspension. MPT was identified as a CsA-sensitive abrupt decrease in light absorbance, reflecting passive matrix swelling.
Assessment of calcein release. Calcein release was monitored in mitochondria using a 96-well fluorometry assay. The mitochondrial pellet was loaded with 1 µM calcein-AM at 37°C (Molecular Probes, USA), washed and submitted to 200 µM CaCl2. At the end of the experiment, mitochondria were centrifuged and calcein fluorescence was determined (Excitation Wavelength: 485 nm, Emission Wavelength: 538 nm) in the pellet (intramitochondrial compartment) and in the supernatant (extramitochondrial compartment). MPT was identified as a CsA-sensitive loss of calcein from the intramitochondrial compartment. This effect was invariably associated with an increase in the calcein at the extramitochondrial compartment.
NMR spectroscopy
Cell pellets ((3–5) x 106 cells per sample) were extracted with 1.2 ml of chloroform–methanol (1: 2) at room temperature for 1 h. After addition of 0.5 ml chloroform and 0.5 ml of water, two phases were separated by centrifugation (1500 g, 5 min). The upper phase (methanol–water) was separated and dried under N2. Dried extracts were dissolved with 400 µl of D2O containing 1 mM 3-(trimethylsilyl) propionic acid (TSP) as chemical shift and concentration reference. Spectroscopy was performed at 27° C with a 400 MHz Bruker ARX spectrometer. Peak areas were measured by integration in fully relaxed spectra.
Statistical analysis
Statistical analysis was performed using commercially available software (SPSS for Windows 8.0). Comparisons between two independent groups were performed by Student's t test. The effect of SI–reoxygenation with or without Gly supplementation on intracellular Gly content was assessed by means of a t test for paired samples. Evaluation of unhomogeneity between multiple groups was performed by analysis of variance (ANOVA). Curves of LDH release were compared by repeated measures ANOVA. Data are expressed as the mean ±S.E.M. The significance level was set at 0.05.
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Results |
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SI did not result in detectable cell death (Fig. 1A). However, reoxygenation after 1 h of SI was associated with a marked increase in the number of PI+ cardiomyocytes, reflecting sarcolemmal disruption (Fig. 1A). Sarcolemmal rupture occurred early upon reoxygenation, was fully observable at 30 min, and did not further increase when the duration of reoxygenation was prolonged to 2 h (data not shown). Apoptosis seemed not to contribute to cell death: the proportion of annexin+–PI– cells after 30 min of reoxygenation was low and remained unchanged after 2 h of reoxygenation (data not shown). Furthermore, this proportion was not significantly modified by either SI or reoxygenation (Fig. 1B).
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Changes in cell volume, intracellular pH and Na+ during SI–reoxygenation
SI induced a significant volume increase (cell swelling), followed by cell size correction upon restoration of energy and normalization of pH. Interestingly, the protective effect of acid pH and Gly on cell death was closely associated with an impaired capacity of cells to recover their volume (Fig. 2A). In both interventions, dose–effect studies demonstrated a narrow inverse relationship between the degree of protection against reoxygenation-induced cell death and the magnitude of correction of cell volume (Fig. 2B and C).
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Reoxygenation induced an important LDH release (Fig. 4A). In hearts submitted to reoxygenation at pH 6.4, total LDH release was reduced by approximately 50% (33.1 ± 6.0 U (g dry tissue)–1 (60 min)–1). Addition of 10 mM Gly during reoxygenation had a similar effect (LDH release was 37.4 ± 7.8 U (g dry tissue)–1 (60 min)–1, Fig. 4B). Normoxic perfusion with a Gly-containing Krebs solution, from 10–4 to 10–1M, did not induce any change in transmembrane action potential characteristics, conduction velocity or contractile function (data not shown).
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In isolated mitochondria, reoxygenation at pH 7.2 after 1 h SI at pH 6.4 was associated with an increase in mitochondrial volume, as measured by a fall in light absorbance. Reoxygenation at pH 6.4 abolished mitochondrial swelling. Reoxygenation-induced swelling can be attributed to MPT since it was blocked by 1 µM CsA and could be precipitated by increasing Ca2+ to 200 µM during reoxygenation (Fig. 5A). MPT was not dependent on substrate availability, since the increase of succinate concentration from 0.3 to 5 mM, or addition of 5 mM pyruvate did not reduce mitochondrial swelling (Fig. 5A). Addition of 3 mM Gly to the reoxygenation buffer completely prevented mitochondrial swelling despite pH normalization (Fig. 5B). This protective effect against MPT could not be mimicked by 5 mML-alanine or by sequestration of extracellular Ca2+ with 2 mM EGTA (Fig. 5B). To further assess the potential inhibitory effect of Gly on MPT, normoxic mitochondria were stressed with 200 µM CaCl2, a manoeuvre known to precipitate mitochondrial permeabilization. The minimum concentration of Gly found to be protective against SI–reoxygenation (3 mM) also reduced mitochondrial swelling induced by high Ca2+ in a similar manner as acidic pH (Fig. 5C and D).
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Addition of 200 µM CaCl2 to calcein-loaded mitochondria induced a rapid and abrupt release of the fluorochrom into the extramitochondrial space (Fig. 6). When Gly was added at the concentrations used to prevent mitochondrial swelling (3 mM or higher), Ca2+-induced calcein release was completely inhibited (Fig. 6). Release of calcein clearly reflected MPT since it could be blocked by CsA (0.1–1 µM).
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NMR spectroscopy revealed that SI–reoxygenation induced a significant reduction of intracellular Gly content. In contrast, a marked increase in intracellular Gly concentration was observed after 30 min reoxygenation when reoxygenation buffer contained 3 mM Gly (Fig. 7).
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Discussion |
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MPT contributes to cardiomyocyte death induced by normalization of pH during reoxygenation
Disruption of the mitochondrial inner membrane by MPT triggers cell death by different pathways, including the release of cytochrome c and other proapoptotic factors that activate caspases and initiate apoptosis (Zamzami & Kroemer, 2001; De Giorgi et al. 2002). Less known, but increasingly recognized, is the contribution of MPT to acute necrotic cell death through impaired ATP synthesis and alterations in cation, in particular of Ca2+, homeostasis (Crompton, 1999; Kim et al. 2003).
Growing evidence indicates that MPT may contribute to myocardial reperfusion or reoxygenation injury. During prolonged ischaemia, the rapid and profound fall in intracellular pH exerts a powerful inhibitory effect on MPT by antagonizing Ca2+ binding to the adenine nucleotide translocase (ANT; Halestrap, 1991). Upon reperfusion, the protective inhibitory effect of low pH is abrogated by the rapid correction of intracellular acidosis, while exacerbated oxidative stress and persistent abnormalities of Ca2+ handling may stimulate MPT. Experimental evidence of MPT during reperfusion has been provided by fluorescence microscopy of cardiomyocytes re-energized after SI showing CsA-sensitive mitochondrial depolarization (Duchen et al. 1993) and calcein loading (Petronilli et al. 1999). In reperfused myocardium, MPT has been demonstrated by the loss of mitochondrial NAD+ (Di Lisa et al. 2001) and mitochondrial entrapment of [3H]deoxyglucose 6-phosphate (Javadov et al. 2000), with a time course that reflects that of pH normalization.
In the present study, acidic reoxygenation markedly attenuated cell death in cultured HL-1 myocytes and in rat hearts. The protective effect of low pH was independent of the inhibitory effect of acidosis on contractility (since HL-1 cells have only a rudimentary contractile machinery and do not hypercontract upon reoxygenation), was not associated with attenuated cytosolic Na+ overload, and was not modified by elimination of extra or intracellular Ca2+. The protective effect of acidic pH cannot be thus explained by attenuation of Ca2+ overload or hypercontracture, but it rather appears to be related to its inhibitory effect on MPT, as observed in isolated mitochondria.
Gly prevents reoxygenation injury by interfering with MPT
The direct protective effect of Gly against necrotic and apoptotic cell death induced by different insults, including ischaemia–reperfusion, and a variety of other pathological conditions – i.e. endotoxaemic shock, alcoholic hepatitis, hepatic fibrosis, arthritis, tumour and drug toxicity – has been characterized in detail (Zhong et al. 1996; Nishimura et al. 1998; Nishimura & Lemasters, 2001; Dong et al. 2001; Zhang et al. 2003). Gly delays sarcolemmal rupture induced by hypoxia, which was initially explained by attenuated Na+ accumulation obscurely related to stimulation of the Gly-gated Cl– channels, originally described in the postsynaptic membranes of the spinal cord (Carini et al. 2000). Other authors suggested that the protective effect of Gly was independent of receptor stimulation but rather related to the closure of non-specific leaks in plasma membranes induced by hypoxia (Dong et al. 1998; Frank et al. 2000; Nishimura & Lemasters, 2001). Confocal microscopy analysis of membrane permeabilization to fluorescent molecules of different sizes demonstrated that Gly delays membrane permeabilization during hypoxia, which could reflect the opening of large anionic channels (‘death pores’) eventually leading to massive cell swelling and sarcolemmal rupture (Nishimura & Lemasters, 2001). Previous studies were coincident in interpreting that Gly prevented plasma membrane failure, the final event in the pathway to necrotic cell death (Frank et al. 2000).
In the present study Gly completely prevented cell death associated with normalization of pH during reoxygenation in cardiomyocyte cultures, and mimicked the protective effect of acidic reoxygenation on LDH release in isolated rat hearts. Since acute cell death observed during reoxygenation in our model was ascribable to MPT, we hypothesized that Gly could act at the mitochondrial level, interfering with MPT. The experiments in isolated mitochondria demonstrated that Gly indeed prevents CsA-sensitive mitochondrial swelling and calcein release induced by re-energization after a transient SI or by exposure to high Ca2+. The protective effect of Gly is not related to its utilization as a respiratory substrate or as a source of NAD(P)H, since it was not mimicked by other substrates (succinate or pyruvate) and was present in fully energized mitochondria submitted to Ca2+ overload.
The inhibitory effect of Gly on MPT was equivalent to that of low pH (6.4) and was observed at high concentrations (3 mM). Intracellular Gly concentration, measured by NMR spectroscopy, was reduced in HL-1 cells exposed to SI–reoxygenation, as compared to normoxic cells. Exposure to 3 mM Gly during reoxygenation raised intracellular Gly concentration up to the range proven to inhibit MPT. Measured intracellular content corresponds to an estimated Gly concentration of approximately 7 mM, compared to approximately 1 mM in normoxic cells and 0.6 mM in cells reoxygenated without Gly.
Hypothetical role of Gly depletion in reoxygenation
The mechanism of Gly depletion in cardiomyocytes submitted to SI–reoxygenation was not investigated in the present study. However, amino acid and Cl– release play an important role in regulating volume decrease in swollen cells (Rasmusson et al. 1993; Song et al. 1998). It should be thus expected that the rapid reduction in cell volume occurring during reoxygenation and normalization of intracellular pH results in Gly efflux. Although the contribution of Gly efflux to volume decrease has not been established, its abolition by exposure to increased Gly concentration in the extracellular medium should reduce it and hamper cell volume correction. In this study, addition of Gly to the reoxygenation buffer attenuated volume decrease, and this effect was closely correlated with the magnitude of the protection afforded against cell death.
A hypothesis that integrates the results obtained in the present study is outlined in Fig. 8. Rapid pH normalization during reoxygenation favours MPT and cell death. Intracellular Gly acts as an inhibitor of MPT. However, volume regulatory decrease occurring upon normalization of extracellular pH involves cellular Gly loss, which makes the cell more vulnerable to MPT, an effect that can be reversed by increasing extracellular Gly concentration.
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Footnotes |
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References |
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Crompton M (1999). The mitochondrial permeability transition pore and its role in cell death. Biochem J 341, 233–249.[CrossRef][Medline]
De Giorgi F, Lartigue L, Bauer MK, Schubert A, Grimm S, Hanson GT, Remington SJ, Youle RJ & Ichas F (2002). The permeability transition pore signals apoptosis by directing Bax translocation and multimerization. FASEB J 16, 607–609.
Di Lisa F, Menabo R, Canton M, Barile M & Bernardi P (2001). Opening of the mitochondrial permeability transition pore causes depletion of mitochondrial and cytosolic NAD+ and is a causative event in the death of myocytes in postischemic reperfusion of the heart. J Biol Chem 276, 2571–2575.
Dong Z, Patel Y, Saikumar P, Weinberg JM & Venkatachalam MA (1998). Development of porous defects in plasma membranes of adenosine triphosphate-depleted Madin-Darby canine kidney cells and its inhibition by glycine. Laboratory Invest 78, 657–668.[Medline]
Dong Z, Venkatachalam MA, Weinberg JM, Saikumar P & Patel Y (2001). Protection of ATP-depleted cells by impermeant strychnine derivatives: implications for glycine cytoprotection. Am J Pathol 158, 1021–1028.
Duchen MR (1999). Contributions of mitochondria to animal physiology: from homeostatic sensor to calcium signalling and cell death. J Physiol 516, 1–17.
Duchen MR, McGuinness O, Brown LA & Crompton M (1993). On the involvement of a cyclosporin A sensitive mitochondrial pore in myocardial reperfusion injury. Cardiovasc Res 27, 1790–1794.
Frank A, Rauen U & de Groot H (2000). Protection by glycine against hypoxic injury of rat hepatocytes: inhibition of ion fluxes through nonspecific leaks. J Hepatol 32, 58–66.[Medline]
Garlid KD, Paucek P, Yarov-Yarovoy V, Sun X & Schindler PA (1996). The mitochondrial KATP channel as a receptor for potassium channel openers. J Biol Chem 271, 8796–8799.
Halestrap AP (1991). Calcium-dependent opening of a non-specific pore in the mitochondrial inner membrane is inhibited at pH values below 7. Implications for the protective effect of low pH against chemical and hypoxic cell damage. Biochem J 278, 715–719.[Medline]
Hausenloy DJ, Duchen MR & Yellon DM (2003). Inhibiting mitochondrial permeability transition pore opening at reperfusion protects against ischaemia-reperfusion injury. Cardiovasc Res 60, 617–625.
Holmuhamedov EL, Jovanovic S, Dzeja PP, Jovanovic A & Terzic A (1998). Mitochondrial ATP-sensitive K+ channels modulate cardiac mitochondrial function. Am J Physiol 275, H1567–H1576.[Medline]
Javadov SA, Clarke S, Das M, Griffiths EJ, Lim KH & Halestrap AP (2003). Ischaemic preconditioning inhibits opening of mitochondrial permeability transition pores in the reperfused rat heart. J Physiol 549, 513–524.
Javadov SA, Lim KH, Kerr PM, Suleiman MS, Angelini GD & Halestrap AP (2000). Protection of hearts from reperfusion injury by propofol is associated with inhibition of the mitochondrial permeability transition. Cardiovasc Res 45, 360–369.
Kim JS, He L & Lemasters JJ (2003). Mitochondrial permeability transition: a common pathway to necrosis and apoptosis. Biochem Biophys Res Commun 304, 463–470.[CrossRef][Medline]
Lemasters JJ, Nieminen AL, Qian T, Trost LC, Elmore SP, Nishimura Y, Crowe RA, Cascio WE, Bradham CA, Brenner DA & Herman B (1998). The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy. Biochim Biophys Acta 1366, 177–196.[Medline]
Nilsson A, Randmaa I & Hahn RG (1996). Haemodynamic effects of irrigating fluids studied by Doppler ultrasonography in volunteers. Br J Urol 77, 541–546.[Medline]
Nishimura Y & Lemasters JJ (2001). Glycine blocks opening of a death channel in cultured hepatic sinusoidal endothelial cells during chemical hypoxia. Cell Death Differ 8, 850–858.[CrossRef][Medline]
Nishimura Y, Romer LH & Lemasters JJ (1998). Mitochondrial dysfunction and cytoskeletal disruption during chemical hypoxia to cultured rat hepatic sinusoidal endothelial cells: the pH paradox and cytoprotection by glucose, acidotic pH, and glycine. Hepatology 27, 1039–1049.[CrossRef][Medline]
Petronilli V, Miotto G, Canton M, Brini M, Colonna R, Bernardi P & Di Lisa F (1999). Transient and long-lasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence. Biophys J 76, 725–734.
Petronilli V, Penzo D, Scorrano L, Bernardi P & Di Lisa F (2001). The mitochondrial permeability transition, release of cytochrome c and cell death. Correlation with the duration of pore openings in situ. J Biol Chem 276, 12030–12034.
Rasmusson RL, Davis DG & Lieberman M (1993). Amino acid loss during volume regulatory decrease in cultured chick heart cells. Am J Physiol 264, C136–C145.[Medline]
Rodriguez-Sinovas A, Garcia-Dorado D, Padilla F, Inserte J, Barrabes JA, Ruiz-Meana M, Agullo L & Soler-Soler J (2003). Pre-treatment with the Na+/H+ exchange inhibitor cariporide delays cell-to-cell electrical uncoupling during myocardial ischemia. Cardiovasc Res 58, 109–117.[CrossRef][Medline]
Ruiz-Meana M, Garcia-Dorado D, Pina P, Inserte J, Agullo L & Soler-Soler J (2003). Cariporide preserves mitochondrial proton gradient and delays ATP depletion in cardiomyocytes during ischemic conditions. Am J Physiol Heart Circ Physiol 285, H999–H1006.
Schemmer P, Bradford BU, Rose ML, Bunzendahl H, Raleigh JA, Lemasters JJ & Thurman RG (1999). Intravenous glycine improves survival in rat liver transplantation. Am J Physiol 276, G924–G932.[Medline]
Song D, O'Regan MH & Phillis JW (1998). Amino acid release during volume regulation by cardiac cells: cellular mechanisms. Eur J Pharmacol 341, 273–280.[CrossRef][Medline]
Zamzami N & Kroemer G (2001). The mitochondrion in apoptosis: how Pandora's box opens. Nat Rev Mol Cell Biol 2, 67–71.[CrossRef][Medline]
Zhang K, Weinberg JM, Venkatachalam MA & Dong Z (2003). Glycine protection of PC-12 cells against injury by ATP-depletion. Neurochem Res 28, 893–901.[CrossRef][Medline]
Zhong Z, Jones S & Thurman RG (1996). Glycine minimizes reperfusion injury in a low-flow, reflow liver perfusion model in the rat. Am J Physiol 270, G332–G338.[Medline]
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