© 2000 by European Society of Cardiology
Copyright © 2000, European Society of Cardiology
Apoptosis induction by nitric oxide in adult cardiomyocytes via cGMP-signaling and its impairment after simulated ischemia
Physiologisches Institut, Justus-Liebig-Universität Giessen, Giessen, Germany
* Corresponding author. Tel.: +49-641-994-7243; fax: +49-641-994-7239 Gerhild.Taimor{at}physiologie.med.uni-giessen.de
Received 2 June 1999; accepted 16 August 1999
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Abstract |
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 Top Abstract 1 Introduction2 Methods3 Results4 DiscussionAcknowledgmentsReferences |
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Objective: Nitric oxide (NO) has been shown to induce apoptosis in cardiomyocytes under normoxic conditions. The ability of NO to induce apoptosis after ischemia–reperfusion, a situation of increased NO release in vivo, has not been investigated. The present study was undertaken to characterize the pathway of induction of apoptosis by NO and the influence of ischemia on this pathway in cardiomyocytes. Methods: The study was performed on isolated adult cardiomyocytes of the rat. Ischemia was simulated by anoxia in a glucose free medium, pH 6.4. Induction of apoptosis was detected (1) by annexinV-fluorescein isothiocyanate (annexinV-FITC) binding to cells under exclusion of propidium iodide and (2) by laddering of genomic DNA. Results: Incubation of cardiomyocytes with the NO-donor (±)-S-nitroso-N-acetylpenicillamine (SNAP, 100 µM) induced apoptosis in 14.1±1.9% of the cells and necrosis in 24.4±4.6%. The induction of apoptosis but not necrosis could be blocked by inhibition of soluble guanylyl cyclase or of protein kinase G. Apoptosis induction was mimicked by incubation of cardiomyocytes with 8-pCPT-cGMP (100 µM, 9.6±0.6% apoptotic cells) or YC-1 (75 µM, 14.6±2.8% apoptotic cells), a direct activator of soluble guanylyl cyclase. After 3 h of anoxia, cardiomyocytes were transiently protected against apoptosis induced by NO, but not by 8-pCPT-cGMP or YC-1 (8.9±0.7% or 13.4±2.4% apoptotic cells). A correlation of the apoptotic response to SNAP or YC-1 with an increased activity of soluble guanylyl cyclase, determined by measurements of intracellular cGMP contents, was found. Conclusions: NO induces apoptosis in a cGMP dependent manner in isolated adult cardiomyocytes whereas induction of necrosis seems cGMP-independent. After simulated in vitro ischemia the activation of soluble guanylyl cyclase by NO is transiently inhibited resulting in a transient anti-apoptotic protection.
KEYWORDS Apoptosis; Cell culture/isolation; Myocytes; Nitric oxide; Second messengers
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionAcknowledgmentsReferences |
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In recent years several studies have demonstrated the appearance of apoptotic cardiomyocytes in ischemic–reperfused myocardium. Apoptosis of cardiomyocytes may be elicited by conditions extrinsic or intrinsic to this cell type. In a previous study we analysed whether energy depletion is a sufficient intrinsic cause for apoptosis induction in cardiomyocytes [1]. To exclude the influence of extrinsic factors, isolated adult cardiomyocytes were used as an experimental model. When the cells were exposed to simulated ischemia–reoxygenation they progressively developed necrosis, but not apoptosis. Those findings indicated that factors extrinsic to the cardiomyocytes contribute to their apoptotic cell death in ischemic–reperfused myocardium. Nitric oxide (NO) is one such possible extrinsic factor. In ischemic–reperfused myocardium it can be released, e.g. by infiltrating macrophages. Indeed, a correlation between the appearance of apoptosis in ischemic–reperfused myocardium and an increased iNOS expression in infiltrating macrophages has been demonstrated by Suzuki et al. [2]. A correlation between apoptosis induction and iNOS expression was also shown for cardiac allograft rejection [3]. In neonatal as well as in adult cardiomyocytes NO-donors, like S-nitrosoglutathione, (±)-S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitroprusside, have been found to induce apoptosis [4,5,1].
We reported previously [1] that NO induces apoptosis under normoxic conditions in isolated adult cardiomyocytes. It is not known (i) whether NO induces apoptosis in cardiomyocytes via its cGMP dependent signaling mechanism or some other radical related mode of action and (ii) whether NO acts as an apoptotic agent also in reperfusion, when the metabolism of cardiomyocytes is altered due to the preceding ischemic conditions. The present study was undertaken to identify the second messengers leading to NO-induced apoptosis in cardiomyocytes and to analyse how ischemic conditions influence this signaling pathway. Again, a model of isolated adult rat cardiomyocytes exposed to simulated ischemia and subsequent reoxygenation was used. Ischemia was simulated by incubating cardiomyocytes in an anoxic glucose-free medium (pH 6.4), an experimental model which has been characterised in detail before [6–8]. To induce apoptosis, the NO-donor SNAP was used [9,10]. Apoptotic cell damage was monitored (i) by the appearance of DNA laddering in agarose gels, and (ii) by the translocation of phosphatidylserine to the outer cell surface as detected by binding of FITC-labeled annexinV under exclusion of the DNA dye propidium iodide.
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2 Methods |
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The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).
2.1 Cell isolation and short-term cultures
Ventricular cardiomyocytes were isolated from 200–250 g male Wistar rats, suspended in basal culture medium and plated on 60-mm culture dishes, which were preincubated overnight with 4% fetal calf serum in medium 199 as previously described [11]. The basal culture medium (CCT) was modified medium 199 including Earle's salts, 2 mM L-carnitine, 5 mM taurine, 100 IU/ml penicillin, 100 µg/ml streptomycin and 10 µM cytosine-β-D-arabinofuranoside (pH 7.4). Three hours after plating the dishes were washed twice with modified phosphate free Tyrode's medium (140 mM NaCl; 3.6 mM KCl; 1.2 mM Mg2SO4; 1 mM CaCl2 and 20 mM N-2-(hydroxyethyl)-piperazine-N'-2-ethansulfonic acid, pH 6.4). As a result of the medium change broken cells were removed resulting in cultures of about 90% quiescent rod-shaped cells on average.
2.2 Experimental protocols
For simulated ischemia, dishes were filled with 1 ml of the modified Tyrode's medium (pH 6.4), gassed with 100% N2 and incubated at 37°C for various times (1, 2 and 3 h) in gas tight chambers in an atmosphere of 100% N2. Reoxygenation was performed by addition of 1 ml CCT medium and incubation of the cells at 37°C with air oxygen. Time matched controls for anoxic incubations were obtained by use of media equilibrated with air instead of N2 and adjusted to pH 7.4 instead of 6.4.
Apoptosis inducing agents were applied under normoxic conditions, immediately after simulated ischemia or after different times of reoxygenation (30, 60 and 90 min) by addition of 10 µl of an appropriate stock solution in DMSO. Control dishes were incubated with vehicle. Fluorescence staining with annexin V-FITC–propidium iodide was done 2 h and DNA extraction was performed 6 h after addition of agent.
2.3 Analysis of genomic DNA
DNA was extracted as described by Tanaka et al. [12] 6 h after agent addition or in time-matched controls. In brief, cardiomyocytes were harvested by centrifugation at 2800 g for 5 min. After resuspension in lysis buffer (100 mM NaCl; 10 mM Tris–HCl; 25 mM ethylenediaminetetraacetic acid (EDTA); 0.5% sodiumdodecyl sulfate, 100 µg/ml proteinase K, pH 8.0) myocytes were incubated for 3 h at 37°C. After phenol–chloroform extraction and ethanol precipitation DNA was dissolved in TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8.0) and incubated with 5 µg/ml DNAse-free RNAse for 2 h at 37°C. Again the DNA was precipitated and resuspended in TE buffer. Concentrations were measured spectrophotometrically. A 5-µg amount of each DNA was electrophoretically separated on 1.5% agarose gels and stained with ethidium bromide.
2.4 AnnexinV–propidium iodide binding assay
AnnexinV-FITC–propidium iodide staining was performed essentially as described by Vermes et al. [13] and Martin et al. [14]. In detail, 10 µl of annexinV-FITC (Boehringer, Ingelheim, Germany) and 1 ng propidium iodide were added to the culture medium (2 ml) 2 h after addition of agent or in time-matched controls. Cultures were then incubated for 10 min at 37°C in the dark before being analysed by fluorescence microscopy. As an early event of apoptosis, cells translocate phosphatidylserine from the inner site of their plasma membrane to the outer surface [15] while the membrane remains physically intact. Apoptotic cells therefore stain with annexinV-FITC, which binds with high affinity to phosphatidylserine and exclude propidium iodide, a DNA dye, as this is unable to pass through the plasma membrane. Necrotic cells have lost the physical integrity of their plasma membrane and therefore stain both with annexin-FITC and propidium iodide. Cells which are neither apoptotic nor necrotic do not stain with either dye. For quantification of apoptosis and necrosis 300 randomly distributed cells were counted in each experiment.
2.5 Determination of cellular cGMP contents
For measurement of cellular cGMP contents cardiomyocytes were preincubated with the phophodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, 1 mM) for 10 min. Then cells were stimulated in the presence of IBMX for 10 min with different agents. Cellular cGMP contents were analysed with the cGMP-(3H)-assay system from Amersham (Braunschweig, Germany) and expressed in relation to cellular protein content.
2.6 Statistics
Data were analysed using Microcal ORIGIN Version 3.5 (Microcal Software, Northampton, MA, USA). Statistical comparisons involving more than two groups were performed by means of one-way ANOVA test followed by t-tests for independent samples. According to Bonferroni method the significance level is adjusted to the number of compared groups, which were always 4 or 5 in this study [16]; therefore a P value of <0.01 was considered to indicate statistical significance. Comparison between two groups were performed by means of t-tests for independent samples with a critical value of P=0.05. Sample size was 3–5 experiments in independent cell preparations. All values are expressed as means±SEM.
2.7 Materials
Medium 199 was obtained from Boehringer (Mannheim, Germany), fetal calf serum from PAA (Linz, Austria), crude collagenase was from Biochrom (Berlin, Germany), KT5823, ODQ, 8-pCPT-cGMP, SNAP and Z-DEVD-FMK was from Calbiochem (Bad Soden, Germany), IBMX and NAP was from Sigma, YC-1 (3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole) (Alexis, Grünberg, Germany), cGMP-(3H)-assay system was from Amersham (Braunschweig, Germany) and annexinV-FITC was from Boehringer.
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3 Results |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionAcknowledgmentsReferences |
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3.1 Apoptosis induction under normoxic conditions
Incubation of cardiomyocytes with the spontaneous NO donor SNAP for 2 h induced apoptosis dose-dependently in cardiomyocyte cultures, reaching a level of 14.1±1.9% apoptotic and 24.4±4.6% necrotic cells at a concentration of 100 µM SNAP. This has to be compared with a background of 4.7±1.3% apoptotic and 9.0±2.8% necrotic cells found in untreated controls which most likely results from the isolation procedure (P<0.01) (Fig. 1a). The inactive analog of SNAP, N-acetylpenicillamine (NAP, 100 µM), which does not release NO, did not change the rate of cell death compared to the controls. These results obtained by annexinV-FITC propidium iodide staining were confirmed by DNA-laddering. This was only observed in experiments in which cardiomyocytes were incubated with 100 µM SNAP (Fig. 1b). In the presence of the caspase-3 inhibitor Z-DEVD-FMK (10 µM), induction of apoptosis by SNAP was abolished (5.3±2.9% apoptotic cells, not significant vs. control). Incubation of cardiomyocytes with the inhibitor of soluble guanylyl cyclase, ODQ (10 µM) or the inhibitor of protein kinase G, KT5823 (10 µM) inhibited entirely the apoptotic response to SNAP (5.4±0.9% and 6.5±1.1% apoptotic cells, respectively; not significant vs. controls) and DNA-laddering was also no longer detectable (Fig. 1a and b). Neither of the inhibitors had influence on the rate of necrotic cell death after SNAP addition (18.5±2.8% necrotic cells in presence of Z-DEVD-FMK, 23.3±2.7% in presence of ODQ and 24.8±2.8% with KT5823, not significant to SNAP-induced necrosis). These findings indicate that NO-induced apoptosis, but not necrosis, is mediated in a cGMP-dependent manner.
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-DNA, HindIII digested. DNA was stained by ethidium bromide.The NO-independent activator of soluble guanylyl cyclase, YC-1 induced apoptosis dose-dependently (Fig. 2). After addition of 75 µM YC-1, 14.6±2.8% apoptotic cardiomyocytes were found (P<0.01 vs. control). Direct activation of protein kinase G by incubation of the cardiomyocytes with 8-pCPT-cGMP also induced apoptosis concentration-dependently, reaching 9.6±0.6% apoptotic cells at a concentration of 100 µM (P<0.01 vs. control) (Fig. 2).
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3.2 cGMP contents under normoxic conditions
To evaluate the activation of guanylyl cyclase, the rise of cGMP contents in cardiomyocytes incubated in the presence of the unspecific inhibitor of phosphodiesterases IBMX (1 mM) was monitored. Under these conditions, addition of 100 µM SNAP increased cellular cGMP contents by 70.4±1.3% within 10 min as compared to time matched controls (P<0.01 vs. control) (Fig. 3). After addition of YC-1 (75 µM) cGMP levels increased by 73.7±1.9% within 10 min (P<0.01 vs. control) (Fig. 3).
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3.3 Apoptosis induction after anoxia
To simulate ischemia, cells were exposed for 3 h to acidotic anoxic conditions. This was followed by 2 h of reoxygenation. Then cardiomyocytes were stained with annexinV-FITC–propidium iodide. As also demonstrated in a previous study [1], anoxia–reoxygenation did not induce apoptosis in isolated cardiomyocytes. The amount of 4.9±0.7% apoptotic cells is the same as 4.8±1.2% apoptotic cells found under normoxic control conditions (Fig. 4). In contrast to its pronounced apoptotic effect under normoxic conditions, addition of the NO-donor SNAP (100 µM) at the beginning of reoxygenation no longer induced apoptotic cell death (4.6±0.7% apoptotic cells; not significant vs. control).
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In an additional set of experiments we investigated if this protection against SNAP induced apoptosis was maintained during longer periods of reoxygenation. In these experiments SNAP was added not immediately at the beginning of reoxygenation but after 30, 60 or 90 min. In each case the annexinV-FITC–propidium iodide assay was performed 2 h thereafter. We found that the protection against apoptosis was preserved for up to 60 min after simulated ischemia (5.8±0.5% apoptotic cells, when SNAP was added after 60 min of reoxygenation, not significant vs. control). Addition of SNAP after 90 min of reoxygenation, however, induced apoptosis (11.3±2.3% apoptotic cells, P<0.01 vs. control).
During the first hour of reoxygenation, cardiomyocytes were protected against apoptosis induced by the NO-donor SNAP but not against apoptosis induced by YC-1 or 8-pCPT-cGMP: Added at the very beginning of reoxygenation after 3 h of anoxia, YC-1 (75 µM) or 8-pCPT-cGMP (100 µM) induced apoptosis in 13.4±2.4 or 8.9±0.7% of the cells, respectively (P<0.01 vs. control) (Fig. 4).
3.4 cGMP contents after anoxia–reoxygenation
To evaluate activation of soluble guanylyl cyclase, cGMP contents were measured in presence of IBMX after an incubation time of 10 min. In contrast to normoxic conditions, addition of SNAP (100 µM) after 3 h of anoxia, directly at the beginning of reoxygenation, did not increase cellular cGMP contents as compared to time-matched controls (Fig. 5). When YC-1 (75 µM) was added at the beginning of reoxygenation, cGMP contents increased by 76±4.2% within 10 min (P<0.01). The suppression of the cGMP response to NO was transient. When cardiomyocytes were exposed to SNAP after 90 min of reoxygenation, cGMP content rose as under normoxic conditions to 174±3.9% (P<0.01 vs. control) (Fig. 5).
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SNAP was also added to cardiomyocytes at the beginning of reoxygenation after only 1 or 2 h of anoxia and the apoptotic response as well as the cGMP contents were measured. Comparison of cGMP contents with the levels of apoptosis found in presence or absence of SNAP or YC-1 under normoxic or anoxic–reoxygenated conditions demonstrate a linear correlation between these parameters (Fig. 6). Induction of apoptosis by SNAP or YC-1 was thus always accompanied by an activation of soluble guanylyl cyclase. Under conditions when incubation with SNAP did not result in a cGMP-increase, there was no induction of apoptosis.
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): (a) control, (b) SNAP, (c) YC-1; directly after anoxia (
): (d) control, (e) 1 h of anoxia+SNAP, (f) 2 h of anoxia+SNAP (g) 3 h of anoxia+SNAP, (h) 3 h of anoxia+YC-1; or after 3 h of anoxia followed by reoxygenation (
): (i) 30 min reoxygenation+SNAP, (j) 90 min reoxygenation+SNAP. The data are means±SE of five independent preparations. |
4 Discussion |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionAcknowledgmentsReferences |
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Nitric oxide, which is found in increased amounts in ischemic–reperfused myocardium, can induce apoptosis in cardiomyocytes under normoxic conditions [4,5,1]. But the effects of NO on cardiomyocytes under post-ischemic conditions are unknown. In the present study the mechanisms of transduction of the apoptotic signal of NO in adult cardiomyocytes and of how this signaling may be altered after cardiomyocytes had been exposed to conditions of simulated ischemia was investigated. The main findings are that (i) NO induces apoptosis, but not necrosis in a cGMP-dependent manner and that (ii) after simulated ischemia, NO becomes transiently unable to activate soluble guanylyl cyclase and thus to induce cGMP-mediated apoptosis in cardiomyocytes. As a result of the latter, cardiomyocytes are transiently protected against NO-mediated apoptosis.
Induction of apoptosis by the NO-donor SNAP was dose-dependent and could be inhibited by the inhibitor of caspase-3. Caspase-3 has been implicated as mediator of apoptosis in a variety of mammalian cell types [17]. Inhibition of soluble guanylyl cyclase by ODQ [18], or of protein kinase G by KT5823 [19] also totally abolished SNAP-induced apoptosis. The NO-independent activator of soluble guanylyl cyclase, YC-1 [20], and the activator of protein kinase G, 8-pCPT-cGMP [21], were able to induce apoptosis in cardiomyocytes. These results show that apoptosis is mediated via a cGMP-dependent pathway. In contrast to the induction of apoptosis, the necrotic response to SNAP is most likely related to toxic NO-derived radicals, since it could not be blocked by the inhibitors of soluble guanylyl cyclase or of protein kinase G.
After exposure to simulated ischemia cardiomyocytes were transiently protected against SNAP-induced apoptosis, for up to 1 h of reoxygenation. During this time of protection addition of SNAP no longer resulted in an activation of soluble guanylyl cyclase, as determined by measurements of intracellular cGMP levels. In contrast to the transient loss of the apoptotic response to SNAP, direct activation of soluble guanylyl cyclase by YC-1 or of protein kinase G by 8-pCPT-cGMP was still able to induce apoptosis after anoxia. These results demonstrate that the transient loss of the effect of SNAP is not due to post-ischemic inactivation of soluble guanylyl cyclase or protein kinase G. They also suggest that absence of the apoptotic response to NO is not due to a lack of metabolic energy, required to drive the apoptotic process. The latter implication is in agreement with previous observations on the same model showing a rapid recovery of the state of energy [6,1]. The fact, that YC-1 remains capable of inducing apoptosis but SNAP does not, indicates that either the interaction of NO with soluble guanylyl cyclase, involving a binding site different to that of YC-1 [22], is impaired or the ability of SNAP to generate NO intracellularly is compromised. It seems possible, e.g., that reoxygenated cardiomyocytes produce oxygen radicals during the early phase of reoxygenation which scavenge NO.
Increases in intracellular cGMP content due to application of SNAP or YC-1 were always accompanied by induction of apoptosis. A linear correlation between cGMP-increase, which is indicative for activation of soluble guanylyl cyclase, and induction of apoptosis was found (Fig. 6). Previous findings of Wu et al. [5] on normoxic neonatal cardiomyocytes are in accordance with these results. These authors demonstrated that ANP induces apoptosis cGMP-dependently and this response could be mimicked by incubation with 8-Br-cGMP. In contrast to this are findings of the same group [4] showing that cytokine-induced apoptosis in neonatal cardiomyocytes can be mediated via NO in a cGMP-independent manner. This discrepancy might be explained by different NO concentrations produced under these conditions or by other cytokine induced mechanisms which interact with the apoptotic pathway.
In vivo the marked increase of NO in ischemic–reperfused myocardium [23] has been shown to contribute to postischemic injury, since pretreatment with inhibitors of NO-synthesis resulted in a reduction of infarct size [24]. This damage might in part result from NO-induced apoptosis. The present study demonstrates that cardiomyocytes possess an endogenous mechanism of protection against NO-induced apoptosis, activated only transiently post ischemia. If this mechanism could be kept active for longer periods of time, an effective reduction of infarct size may be achieved. Therefore further study of this novel protective mechanism seems promising.
Time for primary review 29 days.
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Acknowledgments |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionAcknowledgmentsReferences |
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The authors want to thank Daniela Schreiber for her excellent technical assistance. This study was supported by a Biomed-II project of the European Union. It is part of a thesis submitted by B. Hofstaetter.
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L. Agullo, D. Garcia-Dorado, N. Escalona, J. Inserte, M. Ruiz-Meana, J. A. Barrabes, M. Mirabet, P. Pina, and J. Soler-Soler Hypoxia and acidosis impair cGMP synthesis in microvascular coronary endothelial cells Am J Physiol Heart Circ Physiol, September 1, 2002; 283(3): H917 - H925. [Abstract] [Full Text] [PDF]
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 A. Ramachandran, D. R. Moellering, E. Ceaser, S. Shiva, J. Xu, and V. Darley-Usmar Inhibition of mitochondrial protein synthesis results in increased endothelial cell susceptibility to nitric oxide-induced apoptosis PNAS, May 14, 2002; 99(10): 6643 - 6648. [Abstract] [Full Text] [PDF]
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P. Narayan, R. M. Mentzer Jr., and R. D. Lasley Annexin V staining during reperfusion detects cardiomyocytes with unique properties Am J Physiol Heart Circ Physiol, November 1, 2001; 281(5): H1931 - H1937. [Abstract] [Full Text] [PDF]
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F. Padilla, D. Garcia-Dorado, L. Agullo, J. A Barrabes, J. Inserte, N. Escalona, M. Meyer, M. Mirabet, P. Pina, and J. Soler-Soler Intravenous administration of the natriuretic peptide urodilatin at low doses during coronary reperfusion limits infarct size in anesthetized pigs Cardiovasc Res, August 15, 2001; 51(3): 592 - 600. [Abstract] [Full Text] [PDF]
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 H. H. Wang, A. R. McIntosh, B. B. Hasinoff, E. S. Rector, N. Ahmed, D. M. Nance, and F. W. Orr B16 Melanoma Cell Arrest in the Mouse Liver Induces Nitric Oxide Release and Sinusoidal Cytotoxicity: A Natural Hepatic Defense against Metastasis Cancer Res., October 1, 2000; 60(20): 5862 - 5869. [Abstract] [Full Text]
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G. Taimor Cardiac gap junctions: good or bad? Cardiovasc Res, October 1, 2000; 48(1): 8 - 10. [Full Text] [PDF]
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M. Borgers, L.-M. Voipio-Pulkki, and S. Izumo Apoptosis Cardiovasc Res, February 1, 2000; 45(3): 525 - 527. [Full Text] [PDF]
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