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(Circulation Research. 2000;86:198.)
© 2000 American Heart Association, Inc.

Cellular Biology

Rapid Activation of Neutral Sphingomyelinase by Hypoxia-Reoxygenation of Cardiac Myocytes

Olga M. Hernandez, Daryl J. Discher, Nanette H. Bishopric, Keith A. Webster

From the Department of Molecular and Cellular Pharmacology, University of Miami Medical Center, Fla.

Correspondence to Keith A. Webster, Department of Molecular and Cellular Pharmacology, Rosenstiel Medical Science Building, Room 6038, University of Miami Medical Center, 1600 NW 10th Ave, Miami, FL 33136. E-mail kwebster{at}chroma.med.miami.edu

	Abstract

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Abstract—Elevated levels of oxygen free radicals have been implicated in the pathways of reperfusion injury to myocardial tissue. The targets for free radicals may include specific as well as random intracellular components, and part of the cellular response is the induction of extracellularly activated and stress-activated kinases. The intermediate signals that initiate these stress responses are not known. Here we show that one of the earliest responses of cardiac myocytes to hypoxia and reoxygenation is the activation of neutral sphingomyelinase and accumulation of ceramide. Ceramide increased abruptly after reoxygenation, peaking at 10 minutes with 225±40% of the control level. Neutral sphingomyelinase activity was induced with similar kinetics, and both activities remained elevated for several hours. c-Jun N-terminal kinase (JNK) was also activated within the same time frame. Treatment of cardiac myocytes with extracellular ceramides also activated JNK. Pretreating cells with antioxidants quenched sphingomyelinase activation, ceramide accumulation, and JNK activation. Ceramide did not accumulate in reoxygenated nonmuscle fibroblasts, and JNK was not activated by reoxygenation in these cells. The results identify neutral sphingomyelinase activation as one of the earliest responses of cardiac myocytes to the redox stress imposed by hypoxia-reoxygenation. The results are consistent with a pathway of ceramide-mediated activation of JNK.

Key Words: ceramide • JNK • ischemia/reperfusion • redox • free radical

	Introduction

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Myocardial ischemia is usually associated with coronary artery disease and involves reduced or eliminated blood supply to a region or regions of the myocardium (reviewed in Reference 1¹ ). Reperfusion involves the restoration of this blood flow and can occur spontaneously or can be induced therapeutically by removal of the occlusion. Whereas early reperfusion is the most effective way to reduce ischemic damage, the therapeutic gains are compromised because reperfusion itself initiates a second phase of injury. In animal models of ischemia and reperfusion, processes initiated by reperfusion may cause up to 60% of the total damage to the heart.^{2 3} Reperfusion injury may be reversible or irreversible depending on the severity and duration of the ischemic period (reviewed in References 1 and 4^{1 4} ). Reversible damage includes postischemic arrhythmias and myocardial stunning, both of which may be caused by a combination of free radical attack and transient postischemic calcium overload. Stunning can be attenuated in various animal models by preventing calcium overload or by pretreating the myocardium with antioxidants or OH^– scavengers.^{5 6} Irreversible damage occurs when the ischemic period is extended and severe. In this case, reperfusion propagates the injury by promoting further myocardial cell death through necrosis, apoptosis, and/or oncosis.⁷

The contribution of free radicals to reperfusion damage is well documented, supported both by direct measurements and by the protective effects of antioxidants.^{1 5 8 9} Electron spin resonance spectroscopy indicates that reperfusion causes a burst of free radicals that peaks 2 to 4 minutes after reperfusion and continues for several hours.¹⁰ The source of the free radicals may be the mitochondrial electron transport chain, which is inhibited during ischemia and reactivated by reoxygenation.⁴ Direct intramyocyte targets for free radical oxidations include polyunsaturated fatty acids, membrane phospholipids, and protein sulfhydryl groups.^{1 11 12} The latter may include relatively specific targets such as myofilament proteins, ion transporters, and ß-adrenergic receptors.^{12 13 14} Indirect targets for free radicals include the mitogen-activated protein kinase (MAPK)/extracellular signal–regulated kinase (ERK), p38, and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) signaling cascades, all of which have been shown to be activated by oxidative stress in different models of ischemia and reperfusion.^{15 16 17 18 19} The intermediate signal(s) for the activation of MAPK/JNKs by oxidative stress has not been determined.

We report here that reoxygenation of hypoxic cardiac myocytes mediates a rapid activation of neutral sphingomyelinase (nSMase) and increased intracellular levels of ceramide. This was blocked by pretreatments with antioxidants and preceded the induction of JNK. The results indicate that nSMase activation with subsequent ceramide signaling may be one of the earliest responses of cardiac myocytes to oxidative stress in this model of ischemia-reperfusion.

	Materials and Methods

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Cell Culture, Hypoxia, and Reoxygenation
Methods for primary culture of neonatal rat cardiac myocytes and fibroblasts have been described previously.^{20 21 22} All procedures involving animals were performed in accordance with institutional guidelines. Details of our methods for exposing cells to hypoxia inside an environmental chamber have also been described in detail elsewhere.^{20 21} Cultures exposed only to hypoxia (without reoxygenation) were lysed under hypoxia using ice-cold deoxygenated buffers. For reoxygenation, plates were removed from the chamber and reoxygenated by replacing the medium with oxygenated medium and incubating under 21% O₂, 5% CO₂. In some experiments, N-acetyl cysteine (NAC) or other antioxidants were added to plates under hypoxia from 100x deoxygenated stock solutions.

Ceramide Assays
Ceramide was quantified by a modified sn-1,2-diacylglycerol (DAG) kinase assay.²³ Briefly, lipids were extracted from cell pellets with chloroform/methanol, saponified,²⁴ and resuspended by sonication in a detergent mixture. Samples were incubated for 2 hours in a reaction mix containing bacterial DAG kinase and [-³²P]ATP. [-³²P]-Ceramides were resolved by silica gel, subjected to autoradiography, and quantified by liquid scintillation counting. Ceramides type III and IV, diacylglycerol, and monoacylglycerol, which were also phosphorylated by DAG kinase, were used as standards.

Sphingomyelinase Assays
Sphingomyelinase activities were measured according to previous reports.^{25 26} Briefly, for nSMase, soluble protein cell extracts were incubated in a reaction mix containing 9 nmol sphingomyelin and 20 000 dpm [N-methyl-¹⁴C]sphingomyelin per sample for 30 minutes at 37°C. The [¹⁴C]phosphocholine hydrolyzed by nSMase was extracted from each sample with chloroform:methanol (2:1), and H₂O and quantified by scintillation counting. For the measurement of acidic sphingomyelinase (aSMase) activity, the reactions were conducted at pH 5.0 with 1 mmol/L EDTA instead of 7.4, as described previously.²⁶ All activities were normalized to total cell protein.

Kinase Assays
JNK and MAPK activities in cellular lysates were determined by using recombinant glutathione S-transferase–c-Jun (1–141) or myelin basic protein (MBP) exactly as described previously.¹⁷ Immunoprecipitation of cleared cell lysates with JNK1/JNK2-specific or ERK2-specific polyclonal antibodies (Santa Cruz Biotechnology) was also exactly as described previously.^{17 27 28 29} Immunocomplex kinase assays were performed using 2 µg of purified glutathione S-transferase–c-Jun (1–141) or 20 µg of MBP. ERK and JNK reactions were resolved in 12.5% discontinuous SDS-polyacrylamide gels, dried, and exposed to Kodak X-AR film for autoradiography.

Reagents
Fumonisin B1 and bacterial DAG kinase were from Calbiochem; C2- and C6-ceramides were from Biomol; ceramides type III and IV, diacylglycerol, monoacylglycerol, NAC, -phenyl N-tert-butylnitrone (PBN), and MBP were from Sigma; and [N-methyl-¹⁴C]sphingomyelin and [-³²P]ATP were from Amersham Life Science.

	Results

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Accumulation of Ceramide During Hypoxia-Reoxygenation
Cardiac myocytes were exposed to hypoxia for 24 hours and reoxygenated as described in Materials and Methods. At the time points indicated in Figure 1, cells were lysed and assayed for ceramide. Ceramide levels were 150% of the basal aerobic level after 24 hours of hypoxia. Reoxygenation resulted in a burst of ceramide production, which was apparent within the first 3 minutes, and peaked after 10 minutes, with 225% of the aerobic level. Ceramide levels began to decline after 10 minutes, there was a second smaller peak after 2 hours, and then the levels declined almost to basal after 4 hours. The early accumulation of ceramide is consistent with a response to redox stress caused by reoxygenation. Hydrogen peroxide (0.5 mmol/L) treatment also caused an elevation of ceramide that peaked within 8 minutes at 240% of control and declined rapidly to just above baseline after 30 minutes. Hydrogen peroxide at 0.5 mmol/L was close to the optimal concentration, given that there was less ceramide produced (and lower nSMase activation) with 0.1 or 1.0 mmol/L (data not shown).

View larger version (48K): [in this window] [in a new window]   Figure 1. Ceramide accumulation during hypoxia-reoxygenation. Cardiac myocytes were exposed to 24 hours of hypoxia (95% N2/5% CO2) and reoxygenated for the indicated times or were treated with 0.5 mmol/L H2O2 for the indicated times. Ceramide was radiolabeled as described in Materials and Methods, resolved by thin-layer chromatography, and identified by autoradiography. Radioactivity was normalized to total protein and is expressed as fold aerobic induction. Values are mean±SEM (for hypoxia-reoxygenation, n=10 to 12 per condition; for H2O2, n=4). **P=0.01 vs aerobic sample.

Activation of Neutral but Not aSMase by Reoxygenation
Ceramide can be generated by a de novo pathway involving ceramide synthase or by hydrolysis of sphingomyelin by sphingomyelinases. Pretreatment of cultures with the ceramide synthase inhibitor fumonisin B1 (1 to 10 µmol/L, previously shown to be optimal for inhibiting ceramide synthase)^{30 31} did not decrease ceramide accumulation at any time point, suggesting that de novo synthesis was not required (data not shown). There are 2 major sphingomyelinases in most cell types; aSMase is predominantly lysosomal, and neutral Mg²⁺-dependent sphingomyelinase (nSMase) is present in both membrane-bound and cytosolic compartments.^{32 33 34} Figures 2A and 2B show the activities of nSMase and aSMase in cardiac myocytes after exposure to hypoxia and reoxygenation, as described in Figure 1. nSMase activity was 120% of the aerobic level after 24 hours of hypoxia, and it increased abruptly to 150% of aerobic level 5 to 10 minutes after reoxygenation. The nSMase activity remained high for 2 to 4 hours and then declined. There was a small but reproducible dip in nSMase activity at 3 minutes; we do not know the reason for this. H₂O₂ also caused nSMase activation that peaked within 5 minutes at 150% control and declined below baseline after 30 minutes. The results are consistent with the ceramide results described in Figure 1. There was no significant change in the activity of the aSMase under any condition at any time (Figure 2B). These results are consistent with activated nSMase as the source of ceramide accumulation in reoxygenated cardiac myocytes.

View larger version (30K): [in this window] [in a new window]   Figure 2. nSMase but not aSMase is activated by hypoxia-reoxygenation. Cardiac myocytes were exposed to 24 hours of hypoxia and reoxygenated for the indicated times, or were treated with 0.5 mmol/L hydrogen peroxide as indicated, and lysed in a neutral or acidic buffer. nSMase and aSMase activity was measured as the amount of radiolabeled phosphocholine released from radiolabeled sphingomyelin in 30 minutes in a reaction mixture of acidic or neutral pH, as described in Materials and Methods. A, nSMase. B, aSMase activity relative to air. Data are mean±SEM normalized to protein (for hypoxia-reoxygenation, n=9 per condition; *P=0.05, **P=0.01 vs aerobic sample; for H2O2, n=2).

Ceramide Accumulation and nSMase Are Quenched by NAC Pretreatment
Previous studies in other cell types have shown that oxidative stress can stimulate ceramide production.^{35 36 37 38 39} To determine whether nSMase activation and ceramide accumulation were related to increased oxidative stress during reoxygenation, cardiac myocytes were treated with 50 mmol/L NAC 30 minutes before reoxygenation, and both ceramide and nSMase were measured again as described in Figures 1 and 2. Figure 3 shows that NAC treatment reduced both ceramide generation and nSMase activation at all time points by as much as 80%. The cumulative effect of NAC treatment in reducing ceramide accumulation and nSMase activity at all time points was significant (P<0.01). Interestingly, NAC treatment did not significantly affect aerobic ceramide or nSMase activities.

View larger version (27K): [in this window] [in a new window]   Figure 3. Ceramide accumulation and nSMase activity induced by hypoxia-reoxygenation are quenched by NAC. Samples in hypoxia were treated with vehicle (gray bars) or 50 mmol/L NAC (solid bars) 30 minutes before reoxygenating, or before addition of 1 mmol/L H2O2 (for 10 minutes) and then assayed for ceramide (A) and nSMase (B) activity as described in Figures 1 and 2. Values are mean±SEM (fold aerobic; n=8 per condition).

Activation of JNK by Ceramide
We and others^{15 17} have shown that hypoxia and reoxygenation of cardiac myocytes induces JNK. Ceramide has been shown previously to induce JNK in other cells.⁴⁰ To determine whether ceramide generation after reoxygenation may be involved with JNK activation, we compared the kinetics of JNK activation and ceramide accumulation at early and late time points after reoxygenation. As shown in Figure 4, there was a close correlation; ceramide was elevated slightly earlier than JNK (within 1 minute), and both activities peaked after 10 to 15 minutes and declined thereafter. Both remained elevated for 2 to 4 hours after reoxygenation. Therefore, the kinetics of ceramide accumulation is compatible with a role for ceramide in the activation of JNK.

View larger version (25K): [in this window] [in a new window]   Figure 4. JNK activation parallels ceramide accumulation. A, Ceramide induction (% aerobic). RO indicates reoxygenation. B, JNK activation by hypoxia-reoxygenation; cardiac myocytes exposed to 24 hours hypoxia were reoxygenated for the indicated times and assayed for ceramide (A) or JNK activity (B) as described in Materials and Methods. C, JNK activation; cardiac myocytes were treated for the indicated times with short-chained C2- and C6-ceramides (cer) or the corresponding dihydroceramides, and natural ceramides (types III and IV), and were then assayed for JNK activity (first second, and fourth panels) or ERK activity (third and fifth panels). Each panel in C is representative of 3 separate experiments.

There are no specific inhibitors of nSMase and no agents that specifically block ceramide signaling; therefore, it is not possible to determine directly whether JNK activation by reoxygenation is dependent on ceramide accumulation. If elevated intracellular ceramide is a signal for JNK activation during reoxygenation, then other treatments that increase intramyocyte ceramide should also activate JNK. Therefore, we asked whether treatment with extracellular ceramides activated JNK. Figure 4C shows the responses to treatments with the cell-permeant C2- and C6-ceramides and with natural ceramides (types III and IV). With all ceramide treatments, there was a lag before JNK was activated; this is probably because of the time required for equilibration of the extracellular ceramides, which have poor aqueous solubility and are delivered as DMSO-BSA complexes. Consistent with this, the cell-permeant ceramides (100 µmol/L) activated JNK within 20 minutes, and there was a longer lag time with the less soluble natural ceramides (30 µmol/L). Ceramides did not affect ERK activity (see third and fifth panels in Figure 4C). These results indicate that extracellular ceramides can initiate a pathway for JNK activation in cardiac myocytes and are compatible with a role for ceramide in the reoxygenation-mediated activation of JNK.

Ceramide Accumulation and JNK Are Quenched in Parallel by Antioxidant Pretreatment
Cardiac myocytes were cultured as described in Figure 3 and were pretreated with the indicated antioxidant before reoxygenation, as described for NAC. Cells were harvested at the indicated times for ceramide assays or after 30 minutes for JNK assay as described above. Pretreatment with either PBN (10 mmol/L) or ß-mercaptoethanol (ß-ME; 1 mmol/L) reduced reoxygenation-mediated ceramide increases at all time points, although the inhibition was not complete (Figure 5A). The cumulative effect of antioxidants on the suppression of ceramide production was significant for PBN and ß-ME (P<0.05). Figure 5B shows the effects of antioxidant pretreatment on JNK activity. Again, all treatments reduced reoxygenation-mediated JNK activity by >50% (determined by densitometric analysis of the autoradiograph, not shown).

View larger version (33K): [in this window] [in a new window]   Figure 5. Ceramide accumulation and JNK are quenched by antioxidant pretreatment. Samples in hypoxia (HX) were treated with vehicle (light gray bars), 1 mmol/L ß-ME, (medium gray bars), or 10 mmol/L PBN (solid bars), 30 minutes before reoxygenating as described in Figure 3. Ceramide (A) and JNK (B) activities were measured as described in Figures 1 and 4. Data are mean±SEM (fold aerobic; n=4 per condition); values without error bars are means of 2 determinations. JNK activity was measured 30 minutes after reoxygenation. JNK was repressed by 65% (ß-ME), 65% (NAC), and 70% (PBN); n=2.

Extracellular Ceramides but Not Reoxygenation Induce JNK in Nonmuscle Fibroblasts
We previously reported that JNK was not induced by reoxygenation of nonmuscle cardiac fibroblasts (NMFs), indicating that JNK activation was at least partially specific for cardiac myocytes.¹⁷ To determine whether the absence of JNK activation was associated with a corresponding absence of ceramide accumulation in these cells, we measured and correlated nSMase, ceramide, JNK, and ERK activities after hypoxia and reoxygenation. These results are shown in Figure 6. Figure 6A shows that although ceramide accumulated in NMFs during exposure to hypoxia, there was no subsequent increase after reoxygenation. Therefore, either reoxygenation is less stressful to NMFs or the signaling pathways activated by free radicals are different. This is supported by nSMase measurements in Figure 6B. In contrast to cardiac myocytes, there was no increase of nSMase activity after reoxygenation of NMFs. Figure 6C confirms that JNK was not activated by reoxygenation in NMFs at any time. To confirm that NMFs were responsive to extracellular ceramides, we measured JNK after ceramide addition, as described in Figure 4. JNK was strongly induced in NMFs by all ceramide treatments (Figure 6D). Therefore, the absence of JNK activation by reoxygenation of NMFs correlates with the absence of nSMase activation and ceramide accumulation in these cells. Similarly to the results with cardiac myocytes, ERK activity was not significantly affected by ceramide treatments of NMFs (Figure 6E).

View larger version (20K): [in this window] [in a new window]   Figure 6. JNK is induced in NMFs by extracellular ceramide but not reoxygenation. NMFs exposed to 24 hours of hypoxia (HX) were reoxygenated for the indicated times and then assayed for ceramide (A), nSMase (B), and JNK (C) activity. NMFs were treated for 1 hour with short-chained C2- and C6-ceramides and natural ceramide type III and then assayed for JNK (D) and ERK (E) activity. In panels A and B, values are mean±SEM (fold aerobic; n=8 per condition). Panels C, D, and E are representative of 3 separate experiments.

	Discussion

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We show that ceramide accumulates rapidly in cardiac myocytes exposed to hypoxia and reoxygenation. The peak values of ceramide (>200% of basal levels) are within the range of, or slightly higher than, those shown previously to activate ceramide signaling in other cells treated with cytokines, ionizing radiation, H₂O₂, or growth factor withdrawal (reviewed in Reference 32³² ). Ceramide accumulation was not sensitive to the ceramide synthase inhibitor fumonisin B1 but was paralleled by the activation of nSMase. Both ceramide accumulation and nSMase activation were biphasic, and preincubating cells with antioxidants significantly inhibited the changes. These results are consistent with a pathway of nSMase activation initiated by redox stress, perhaps involving reactive oxygen. The biphasic nature of nSMase activity and ceramide production is also consistent with the positive feedback control of nSMase by ceramide reported previously.⁴¹ Ceramide has also been shown to interact directly with the mitochondrial electron transport chain generating H₂O₂, which could create another positive feedback loop.^{42 43 44}

This is the first study to demonstrate that hypoxia and reoxygenation activate nSMase and cause ceramide accumulation. Activation of this pathway has been described in other cell types treated with cytokines (tumor necrosis factor [TNF]–), ionizing radiation, and H₂O₂ (reviewed in Reference 32³² ). Therefore, nSMase appears to be activated by multiple stimuli associated with oxidative stress. The molecular mechanisms for this regulation of nSMase activity are not understood; in the case of stimulation with TNF-, nSMase may be activated directly by binding to the TNF- receptor⁴⁵ ; other studies indicate that nSMase is negatively regulated by glutathione.^{46 47 48} In our studies, nSMase was activated in 2 steps, first, during chronic hypoxia, and second, immediately after reoxygenation. Glutathione levels fall precipitously during hypoxia^{17 48} ; therefore, the hypoxia phase of nSMase activation may involve release of glutathione inhibition. The rapid kinetics of nSMase activation during reoxygenation, as well as the responses to antioxidants, is consistent with an upstream role of oxygen free radicals as the initiators. The mechanism of this initiation is not known, but presumably it could involve positive redox regulation of nSMase or negative regulation of an inhibitor.

Previous studies have shown that ceramide can activate JNK through pathways involving TGF-ß–activated kinase-1 or RAS.^{28 32 40} In our studies, the first peak of ceramide accumulation after reoxygenation coincided with the initiation of JNK activity. Activated JNK was detected 8 minutes after reoxygenation and peaked at 15 minutes. JNK activity remained elevated over the basal activity for at least 4 hours. The correlation of ceramide accumulation with JNK activation is consistent with a possible cause-and-effect relationship. Smaller inductions of ceramide than those reported here have been shown to strongly activate JNK and apoptosis in other cell types.⁴⁹ Such a functional relationship between ceramide accumulation and JNK activation is supported by the observation that treatments with cell-permeant and natural ceramides activated JNK in cardiac myocytes. nSMase activity, ceramide accumulation, and JNK activation were all strongly inhibited by NAC and other antioxidants, supporting a common pathway, and neither nSMase nor JNK was induced in reoxygenated nonmuscle fibroblasts. JNK was not strongly induced by hypoxia alone in either cardiac myocytes or NMFs, even though the ceramide level increased. The reason for this is not clear, but it may be related to the more gradual nature of ceramide accumulation during hypoxia, the site of production and compartmentalization of ceramide,⁵⁰ and the acute nature of JNK activation.⁵¹ Alternatively, there may be a moderate, barely detectable but chronic activation of JNK during hypoxia, which would be consistent with the elevated levels of c-Jun seen under these conditions.²¹ Therefore, although our data support a role for ceramide in the activation of JNK by reoxygenation (and this is consistent with previous results), confirmation of such a role will require further experiments. Furthermore, increased ceramide may not be the only stimulus to activate JNK under these conditions. Another pathway has been proposed involving the oxidative inactivation of specific JNK phosphatases.⁵² Although the involvement of this pathway is also speculative, such activity may contribute to the activation of JNK by oxidative stress described here.

H₂O₂ at a concentration of 0.5 mmol/L quantitatively mimicked the effects of reoxygenation. Both nSMase activity and ceramide accumulation peaked early, within 10 minutes of H₂O₂ treatment, and then declined. The relatively longer-lasting effects of hypoxia-reoxygenation compared with H₂O₂ may be related to the reduced levels of glutathione, caused by hypoxia, that only recover gradually after reoxygenation, and as a consequence, may prolong the oxidative stress.¹⁷ Despite their similar effects on nSMase and ceramide, reoxygenation and H₂O₂ may have differential effects on MAPKs. H₂O₂ was previously reported to induce all MAPKs in cardiac myocytes, as well as other cells, with an early activation of p38 followed by slightly later activation of ERK and JNK/SAPK.^{18 52 53 54 55} Hypoxia-reoxygenation, on the other hand, causes strong activation of JNK, but modest or no activation of ERK and p38.¹⁷ The differences may be related to the different sites and species of free radicals generated by these stimuli.^{32 56} Ceramide has also been reported to preferentially induce JNK over ERK in other cells,⁵⁷ and reperfusion causes preferential activation of JNK over ERK in whole-heart preparations.¹⁸ Although these observations are consistent with ceramide-mediated activation of JNK by redox stress, other pathways must also be involved, because H₂O₂, but not ceramide, activates ERK and p38.

Ceramide is the central molecule in the highly conserved sphingomyelin signaling system and serves as a second messenger for numerous cellular functions ranging from proliferation and differentiation to growth arrest and apoptosis (reviewed in References 32 and 56^{32 56} ). Increased tissue ceramide has been reported in reperfused rat hearts.⁵⁸ Alterations of ceramide and JNK activities have been associated with ischemia and reperfusion of both kidney and heart, and may be involved in apoptosis pathways.^{58 59 60 61} Previous studies have shown ceramide accumulation at late stages of apoptosis in cardiac myocytes subjected to hypoxia and metabolic inhibition.⁵⁸ Other studies suggest that the sites of generation and cellular compartmentation determine cellular responses to ceramide changes⁵⁰ and that increased aSMase activity is linked to apoptosis, whereas nSMase activation favors cell survival.^{62 63} Under the conditions of hypoxia-reoxygenation described here, 30% of the cardiac myocytes die by apoptosis after 16 to 20 hours of reoxygenation, whereas 70% survive.^{17 64} The contributions of nSMase activation, increased ceramide generation, and JNK activity to these events are not known.

	Acknowledgments

 
This work was supported by NIH Grant HL44578 (to K.A.W.), by NIH Training Grant T-32 HL07188, and by an Established Investigator Award from the American Heart Association (to N.H.B.).

Received August 23, 1999; accepted October 27, 1999.

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