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J Thorac Cardiovasc Surg 1998;115:210-214
© 1998 Mosby, Inc.
CARDIOPULMONARY SUPPORT AND PHYSIOLOGY |
From the Division of Cardiovascular Surgery, Department of ClinicalBiochemistry and the Centre for Cardiovascular Research, The Toronto Hospitaland the University of Toronto, Toronto, Ontario, Canada. Supported by theHeart and Stroke Foundation of Ontario (grant T2683). V.R., J.S.I.. and F.M.are Research Fellows of the HSFO, R.D.W. is a Career Investigator of the HSFO,and R.K.L. is a Research Scholar of the HSFO.
Received for publication March 11, 1997; revisions requested May14, 1997; revisions received June 6, 1997; accepted for publication August19, 1997. Address for reprints: Richard D. Weisel, MD, EN 14–215, TheToronto Hospial, 200 Elizabeth St., Toronto, Ontario, Canada M5G 2C4.
Abstract
Background: The effects of simulated "ischemia"and "reperfusion" were evaluated in cell cultures of human ventricularcardiomyocytes and human saphenous vein endothelial cells.
Methods: Myocyte and endothelial cell cultures were exposed toa low volume (1.5 ml) of either hypoxic (oxygen tension = 16 mm Hg) oranoxic (oxygen tension = 0 mm Hg) phosphate-buffered saline solutionfor 90 minutes ("ischemia") followed by 30 minutes of simulated "reperfusion."Cell injury was evaluated by trypan blue exclusion. Next, the effects of apreconditioning stimulus were evaluated by a brief (10 minute) exposure tohypoxic or anoxic ischemia and 10 minutes of reperfusion before prolonged(90 minutes) anoxic ischemia. Finally, the effects of anoxic preconditioningon intracellular lactate accumulation and extracellular lactate and acid releasewere assessed.
Results: "Ischemia"and "reperfusion" resulted in greater injury to endothelial cellsthan to cardiomyocytes. In both cell types, anoxic ischemia resulted in greaterinjury than hypoxic ischemia. Preconditioning reduced cell injury in myocytesbut not in endothelial cells. Endothelial cells produced more lactate thancardiomyocytes under normoxic conditions. Ischemia increased lactate accumulationand release in cardiomyocytes but not endothelial cells. Preconditioning reducedlactate accumulation and release in cardiomyocytes but not endothelial cells.
Conclusions: Endothelial cells were more susceptibleto the same period of simulated ischemia than cardiomyocytes. Preconditioningprotected cardiomyocytes but not endothelial cells from a subsequent prolongedperiod of ischemia and reperfusion.
The response of the coronary endothelium to ischemia and reperfusionremains controversial. Morphologic studies
1 have suggested minor changes to the endothelium despite significantischemic changes in myocytes after a coronary occlusion. However, the endotheliumis metabolically active and produces endothelin, 2 nitric oxide, 3 and adenosine. 4 Thesecretion of these and other substances controls coronary perfusion and mayalter myocardial contractility. 5 Ischemic injury to the endothelium may alter its metabolic activitywithout substantially altering structure. Several studies have implicatedendothelial dysfunction in the pathogenesis of myocardial infarction. 6,9 Therefore protection against ischemic injury should be directed towardboth myocytes and endothelial cells.
Ischemic preconditioning is a powerful endogenous mechanism of cardioprotection. 10 Preconditioning has been shownto reduce infarct size by at least 80%. However, the effects of ischemic preconditioningon endothelial cells remains controversial. Recent studies suggest that preconditioningpreserves endothelium-dependent regulation of coronary blood flow. 11,12 Most studies have used whole heart preparations. The differentialeffects of ischemia and the potential benefits of preconditioning have beenincompletely investigated in isolated cell preparations. In addition, studiesevaluating a preconditioning response in human beings did not distinguishbetween the effects on endothelial cells and cardiomyocytes. 13,14
We developed a model of simulated "ischemia" in monolayersof cultured human cells. 15Cultured cells have distinct advantages and definite disadvantages. Cell culturespermit a comparison of isolated cell types to a standard ischemic insult.Therefore the effects of preconditioning on isolated cell cultures permita comparison independent of the influences of other cell types or organ systems.However, the response in cell cultures may not predict the response of wholeorgans to ischemia. In addition, culturing cardiomyocytes and endothelialcells results in cellular dedifferentiation with a loss of some cell functions.We have documented the phenotypic changes that occur with passaging in ourhuman ventricular cardiomyocyte cultures. 16 Although endothelial cells from different vascular beds display differentproperties in vivo, we have found that cultured endothelial cells partiallydedifferentiate to a common cell line. 17 Despite these limitations, we believe that our "ischemic"model provides a unique opportunity to investigate the effects of preconditioningand to explain the differential response of human cardiomyocytes and endothelialcells to simulated "ischemia" and "reperfusion." Inaddition, this model enables the evaluation of pharmacologic agents intendedto mimic the preconditioning response. Agents that have beneficial effectson both cardiomyocytes and endothelial cells may be ideal additives to cardioplegicformulations.
Materials and methods
Human ventricular cardiomyocyte culture.
Cultures of human ventricular cardiomyocytes were established as previouslydescribed. 16,17 In brief, 20 mg biopsy specimens were obtainedfrom the right ventricle of patients undergoing corrective repair of tetralogyof Fallot. The myocardial biopsy specimens were washed in phosphate-bufferedsaline solution (PBS) without calcium. After removal of connective tissue,the myocytes were separated with the use of enzymatic digestion with a mixtureof 0.2% trypsin (Difco Laboratories, Detroit, Mich.) and 0.1% collagenase(Worthington Biochemical Corp., Freehold, N.J.). The isolated cells were culturedat 37° C in 5% carbon dioxide and 95% air in Iscove's modified Dulbecco'smedium (Gibco Laboratories, Grand Island, N.Y.) containing 10% fetal bovineserum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.1 mmol/L ß-mercaptoethanol.Purification was achieved by means of the dilution cloning technique. 16 All cells on the plate were allowedto divide and at 7 days cardiomyocyte, fibroblast, and endothelial coloniesformed. Single cardiomyocyte colonies were then transferred by means of aPasteur pipette to another culture dish. Cell cultures were inspected daily,and any contaminated culture dishes were discarded. Culture purity greaterthan 95% was demonstrated after the third cell passage with fluorescent monoclonalantibody staining for human ventricular myosin heavy chain (Rougier Bio-TechLtd., Montreal, Quebec). Cells passaged four to seven times with a time fromprimary culture of less than 60 days were used for this study.
Human endothelial cell culture.
The technique used to isolate and culture human saphenous vein endothelialcells has been previously reported. 17 In brief, saphenous vein segments were obtained from patients undergoingaorta-coronary bypass operations and washed with PBS to remove blood. Afterremoval of connective tissue, the vein was incubated in an enzymatic solution(0.2% trypsin and 0.1% collagenase in PBS without calcium). Endothelial cellswere isolated by gently washing the inside of the vein segment with culturemedium (medium 199; Gibco) three to five times. The isolated cells were culturedat 37° C in 5% carbon dioxide and 95% air in medium 199 containing 20%fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin.Endothelial cells were distinguished from fibroblasts and smooth muscle cellsby morphologic criteria. The cell culture plates were inspected daily, andany contaminated plates were discarded. Endothelial cell purity was greaterthan 95% in all plates used for these studies and was confirmed by immunofluorescentstaining with a monoclonal antibody against factor VIII antigen. Endothelialcells passaged four to seven times and aged less than 60 days from the timeof primary culture were used for this study.
Experimental protocols.
The cells were studied in PBS with magnesium, calcium, and glucose (MgCl2, 0.49 mmol/L; CaCl2, 0.69 mmol/L; glucose, 3 mmol/L). Ourin vitro technique to simulate "ischemia" and "reperfusion"has been previously described in detail. 15 After 30 minutes of stabilization in 15 ml of normoxic (oxygen tension[Po2] = 150 mm Hg) PBS, "ischemia"was simulated by placing the cells into a sealed acrylic plastic chamber flushedwith 100% nitrogen and exposing the cells to a low volume (1.5 ml) of deoxygenatedPBS for 90 minutes. "Reperfusion" was accomplished by exposureto 15 ml of normoxic PBS for 30 minutes. A small sample of deoxygenated PBS(2 ml) was placed into a center dish in the chamber to monitor temperatureand to confirm the absence of oxygen at the end of the ischemic period. Thetemperature was maintained at 37° C throughout the experiment.
Fig. 1 summarizes the experimental protocols. Cells were assessed after 30 minutesof stabilization, 90 minutes of prolonged "ischemia" (I), and90 minutes of prolonged "ischemia" followed by 30 minutes of "reperfusion"(IR). Two different levels of deoxygenated PBS were used: anoxic (Po2 = 0 mm Hg)and hypoxic (Po2 = 16 mm Hg).
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Assessment of cellular injury.
Cellular injury was assessed with the use of nonconfluent cultures ofcardiomyocytes and endothelial cells (approximately 337,000 cells per 9 cmdiameter culture dish). After the intervention of interest, cell plates wereincubated with 0.4% trypan blue dye (Sigma Chemical Company, St. Louis, Mo.),dissolved in normal saline solution, and assessed for injury under an invertedlight microscope (Nikon Canada Instrument Inc., Mississauga, Ontario) at 200xmagnification. Injured cells were unable to exclude the large molecular weightdye and stained blue. The number of blue-stained cells was counted from fivestandard locations on each plate and expressed as a percentage of the totalnumber of cells. All counts were performed by a single, blinded observer.
Biochemical measurements.
Confluent cultures of cardiomyocytes and endothelial cells (approximately600,000 cells per culture dish) were used for biochemical analysis. Afterremoval from the culture dish, the cardiomyocytes, endothelial cells, andthe extracellular fluid recovered from each intervention were analyzed forlactate by means of an enzymatic method (Stat-Pack rapid lactate test kit,Behring Diagnostics, La Jolla, Calif.). The concentration of hydrogen ion[H+] in the extracellular fluid was determined by converting thepH value measured with a blood gas analyzer (1312 Blood Gas Manager, InstrumentationLaboratory, Milan, Italy) to [H+] by the formula: [H+]= Antilog (–pH). The DNA in the extract of cells was recovered in 5%perchloric acid and quantitated with the use of a spectrophotometric, diphenylaminecolor reaction with calf thymus DNA as the standard. Hydrogen ion and lactatevalues were then corrected for DNA content from each plate.
Statistical analysis.
The SAS program (SAS Institute, Cary, N.C.) was used for analysis. Resultsare expressed as the mean ± standard error with eight plates pergroup. A two-way analysis of variance (ANOVA) was used to test the main effects: cell type (cardiomyocytes or endothelial cells), intervention (ischemia and reperfusion), Po2 (0 or 16 mm Hg), and preconditioning(with or without preconditioning) plus their interactive effects. The factorialdesign of the study permits a complete statistical evaluation by a two-wayANOVA without multiple comparisons between groups. When statistically significantdifferences were found, they were specified by a one-way ANOVA and Duncan'smultiple range test. Statistical significance was assumed at p < 0.05.
Results
Cell injury assessment.
Cellular injury as assessed by trypan blue uptake increased after "ischemia"and "reperfusion" (intervention, F= 188, p < 0.0001). Reperfusion resultedin further injury to cardiomyocytes but not endothelial cells (intervention·celltype, F = 11, p< 0.0001) using both anoxic and hypoxic PBS (intervention·cell type·Po2, F = 7, p =0.002; p < 0.05 for cardiomyocytesand p > 0.05 for endothelial cells byDuncan's multiple range test). Cellular injury was significantly greater overallfor endothelial cells than cardiomyocytes (cell type, F = 38, p < 0.0001), butthe difference was significant at a Po2 of 0 mm Hg and not at a Po2of 16 mm Hg.
Cellular injury increased in both endothelial cells and cardiomyocytesas the Po2 fell from hypoxic (16mm Hg) to anoxic (0 mm Hg) levels (Po2, F = 54, p < 0.0001). Reperfusion after hypoxia resulted in greaterinjury to cardiomyocytes but not endothelial cells (intervention·celltype·Po2, F = 7, p = 0.0002; p < 0.05 for cardiomyocytes, p > 0.05 for endothelial cells by Duncan's multiple rangetest). The effects of "ischemia" and "reperfusion"are summarized in Fig. 2.
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Figs. 3 and 4.
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Discussion
Human cell cultures.
The cardiomyocytes and endothelial cells used in these studies havebeen extensively evaluated in previous reports. 15-19 The endothelial cells isolatedfrom human saphenous veins retain their histologic characteristics after fourto seven passages. In addition, they retain their antioxidant enzyme activityand are readily stained for factor VIII. 17 We found that endothelial cells from different vascular beds becomeindistinguishable after isolation and culture. Thus we believe that thesecells are a useful model of human endothelium but may not reflect the in vivoresponses of coronary endothelium.
The cardiomyocytes retain many similarities to freshly isolated cellsbut also have distinct differences. After enzymatic digestion and passaging,the cardiomyocytes become quiescent. Other investigators have reported thatadult rat cardiomyocytes become quiescent once cultured. 20,21 Althoughthe cardiomyocytes retain an abundant supply of mitochondria and contractileproteins, the sarcomeres become disrupted during division and do not reestablishtheir characteristic functional format. The effects of culturing and passagingthese cells has been extensively described in a previous publication. 16 Although the biochemical and molecularproperties of these cells resemble in vivo cardiomyocytes, these cells undergoa partial phenotypic change, become quiescent, and regain their ability todivide. Other investigators have shown that senescent rat cardiomyocytes mayalso regain their ability to divide in culture. 21,22 Therefore, despite their quiescentstate, we believe that these cells are phenotypically cardiomyocytes and retainmany characteristics of normal human myocardium and may simulate the humanheart during cardioplegic arrest. Thus these cells provide a unique opportunityto evaluate the cellular response to "ischemia," "reperfusion,"and preconditioning.
Simulated "ischemia" and "reperfusion."
Exposing the cells to 90 minutes of a solution with an oxygen contentof either 0 or 16 mm Hg injured both cardiomyocytes and the endothelial cells.Reducing the volume of solution over the cells from 15 to 1.5 ml resultedin the accumulation of the products of ischemic metabolism and a marked reductionin the extracellular pH. Therefore this model is similar to the effects ofglobal ischemia on the heart. Unfortunately, the volume overlying the cellsremains greater than the solution to which the cells are exposed during globalischemia. Our model differs from simple hypoxia and reperfusion as a resultof the reduction in supernatant volume. If cells are exposed to 15 ml of anoxicPBS, the degree of metabolite accumulation and cellular injury is markedlyreduced. 19 Thus we believethat this model may serve as a useful tool to investigate the cellular responseto ischemic preconditioning.
Susceptibility to "ischemia."
We found that endothelial cells were more susceptible to simulated ischemiaunder both anoxic (Po2 = 0 mm Hg)and hypoxic (Po2 = 16 mm Hg) conditions.Previous studies examining endothelial susceptibility to ischemia were contradictory. 6-9,23-27 Both histologic and ultrastructural examination reveal very littleendothelial cell injury during global cardiac ischemia or after reperfusion. 1 The lack of histologic abnormalitiesin endothelial cells despite extensive myocyte damage led to the conclusionthat endothelial cells were resistant to ischemia. Studies on the effectsof ischemia and reperfusion on endothelial function have suggested that thesecells may be as susceptible as cardiomyocytes to ischemic injury. Hulsmannand Dubelaar 23 found thatendothelial dysfunction was substantial after brief ischemic episodes. Similarly,Nakanishi and colleagues 6found severe endothelial dysfunction after global myocardial ischemia andreperfusion and a significant impairment of endothelium-dependent coronaryvascular relaxation. In contrast, Tanaka and colleagues 24 demonstrated that cardiomyocytes were more sensitiveto ischemia than cultures of nonmyocytes. These investigators found DNA fragmentationin neonatal rat cardiomyocytes exposed to 12 hours of hypoxia, whereas thenonmyocyte cultures did not display DNA fragmentation for up to 72 hours.Unfortunately, these authors did not characterize the population of theirnonmyocyte cells.
During whole heart ischemia, endothelial cells may be spared the lowestPo2s because of their perfusion bycollateral circulation. Therefore the relatively sparse histologic injurymay be related to the lesser degree of hypoxia. In this study we found thata Po2 of 16 mm Hg was sufficientto significantly reduce cellular injury compared with anoxic (Po2 = 0 mm Hg) ischemia.
Reperfusion.
Most studies have suggested that endothelial dysfunction occurs duringreperfusion. 6–9 However, we found that reperfusion did notincrease injury in endothelial cells after either hypoxic or anoxic ischemia.In contrast, cardiomyocytes displayed greater injury after reperfusion atboth levels of ischemic insult. Our technique of assessing cell viabilityrequires 5 minutes of incubation in trypan blue. It is possible that thisbrief period of reperfusion after ischemia was sufficient to cause reperfusioninjury in endothelial cells, which was not exacerbated with an additional30 minutes of reperfusion. After anoxic ischemia and reperfusion, cellularinjury was greater in endothelial cells than in cardiomyocytes. The noncontractilenature of the cardiomyocytes may have been protective against ischemia. Energy-consumingcontraction during reperfusion may exacerbate cardiomyocyte injury to a greaterdegree than observed in this study.
Ischemic preconditioning.
Ischemic preconditioning was first described by Murry, Jennings, andReimer 10 in 1986 and mayrepresent the most potent form of endogenous myocardial protection. Althoughthe effects of preconditioning on cardiomyocyte viability is well established, 18,19 the ability of endothelial cells to exhibit a preconditioned responseremains controversial. Defily and Chilian 11 reported that ischemic preconditioning preserved endothelium-dependentcoronary vasodilatation during subsequent prolonged ischemia and reperfusion.They speculated that the preservation of vascular function facilitated myocardialmyocyte preservation. With a similar canine coronary occlusion model, Bauerand colleagues 12 reportedthe opposite results. They concluded that the effect of ischemic preconditioningdid not extend to the coronary vasculature. Endothelial cell viability wasnot evaluated in either study. Most studies on ischemic preconditioning havedemonstrated that cardiomyocyte viability is preserved, but the effects onventricular function remain controversial. 25,26 Therefore it is possible thatischemic preconditioning could affect the metabolic activity of the endothelialcells without preserving cell viability.
Our previous studies have suggested that cardiomyocyte preconditioningis mediated by adenosine receptor stimulation and involves activation of proteinkinase C. 18,19 It is possible that these mechanisms are not active in endothelialcells, which may explain the lack of a protective effect in response to apreconditioning stimulus. Adenosine released from endothelium can be rapidlydeaminated. 27,28 Therefore receptor activation could be preventeddespite adenosine release into the culture medium. Endothelial cells are knownto have adenosine A2 receptors, but adenosine A1 andperhaps A3 receptors are required for ischemic preconditioning.The activity of G proteins, cyclic adenosine monophosphate, and adenosinetriphosphate–sensitive potassium channels in endothelial cells has notbeen well established. Therefore it is possible that the mechanisms requiredfor ischemic preconditioning may not be available in endothelial cells.
We found that a Po2 of 16 mmHg resulted in mild injury to both cardiomyocytes and endothelial cells comparedwith a Po2 of 0 mm Hg. In addition,we demonstrated that hypoxia did induce a preconditioning effect in cardiomyocytes,which was not as potent as that obtained with an anoxic stimulus. If the releaseof adenosine contributes to the preconditioning effect, it is possible thathypoxia results in less adenosine accumulation with a resultant diminutionin the protective response. We were surprised to find that reperfusion afterhypoxic preconditioning and ischemia augmented endothelial injury. This effectmay represent the culmination of three separate insults: a brief period ofischemia, prolonged ischemia, and reperfusion. In endothelial cells exposedto anoxic ischemia, we observed approximately 50% cellular injury. Reperfusionafter hypoxic preconditioning and ischemia also resulted in 50% cellular injury.Therefore at Po2s of 16 mm Hg, weobserved incremental cell injury after the preconditioning stimulus, prolongedischemia, and reperfusion. At Po2sof 0 mm Hg, 50% cellular injury was observed after ischemia alone, and reperfusiondid not result in further injury. It is possible that cells capable of survivinganoxic ischemia were resistant to reperfusion injury, whereas cells mildlyinjured by hypoxic ischemia suffered augmentation of their injury after reperfusion.Nakanishi and associates 6also found that reperfusion was required to demonstrate endothelial dysfunctionafter ischemia. These authors were unable to demonstrate an impairment ofendothelium-dependent coronary vascular relaxation after ischemia alone. Theyused a canine model with abundant coronary collateral circulation, which mayallow for modest Po2s resemblingour hypoxic conditions.
Glycolysis.
We demonstrated that endothelial cells produce more lactate but maintaina normal pH under normoxic conditions of stabilization or reperfusion. Mertensand colleagues 29 found thesame phenomena in rat coronary endothelial cells. The amount of lactate producedwas greater than that reported by Spahr and colleagues, 30 who studied cultured rat cardiomyocytes. During90 minutes of ischemia, cardiomyocytes increased both intracellular and extracellularlactate. Endothelial cells did not increase anaerobic glycolysis during prolongedischemia. Preconditioning significantly reduced lactate release from cardiomyocytes.Endothelial cell lactate accumulation was not affected by preconditioning.The suppression of glycolysis by cardiomyocytes has been previously reportedin cardiomyocyte cell cultures, 18 and this phenomenon has been reported in vivo by Murry and colleagues. 31
In summary, these investigations permit the following conclusions:
1. Endothelial cells were more susceptible to simulated ischemia andreperfusion than cardiomyocytes.
2. Reperfusion increased injury in cardiomyocytes but not endothelialcells.
3. Ischemic preconditioning reduced cell injury in cardiomyocytes butnot endothelial cells.
4. Ischemic preconditioning diminished lactate production in cardiomyocytesbut not endothelial cells.
We believe that attempts to mimic the preconditioning response by pharmacologicadditives to cardioplegic formulations must address the differential requirementsof both cardiomyocytes and endothelial cells. Furthermore, consideration mustbe given to additives that provide beneficial effects on both viability andfunction.
We acknowledge the statistical assistance of Ms. Joan Ivanov, RN, MSc,for her contributions to this manuscript.
References
This article has been cited by other articles:
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  D. M. YELLON and J. M. DOWNEY Preconditioning the Myocardium: From Cellular Physiology to Clinical Cardiology Physiol Rev, October 1, 2003; 83(4): 1113 - 1151. [Abstract] [Full Text] [PDF]
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 N. Shiono, V. Rao, R. D. Weisel, M. Kawasaki, R.-K. Li, D. A. G. Mickle, P. W. M. Fedak, L. C. Tumiati, L. Ko, and S. Verma L-Arginine protects human heart cells from low-volume anoxia and reoxygenation Am J Physiol Heart Circ Physiol, March 1, 2002; 282(3): H805 - H815. [Abstract] [Full Text] [PDF]
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 Y.-F. Chen, S.-C. Wu, C.-H. Huang, P.-C. Pan, C.-S. Lee, and C.-C. Lin Morphometric identification of luminal narrowing of myocardial capillaries after cardioplegic arrest Ann. Thorac. Surg., January 1, 2001; 71(1): 243 - 248. [Abstract] [Full Text] [PDF]
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 E. Kevelaitis, A. Oubenaissa, C. Mouas, J. Peynet, and P. Menasche Ischemic preconditioning with opening of mitochondrial adenosine triphosphate-sensitive potassium channels or Na+/H+ exchange inhibition: Which is the best protective strategy for heart transplants? J. Thorac. Cardiovasc. Surg., January 1, 2001; 121(1): 0155 - 162. [Abstract] [Full Text] [PDF]
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 A. T. SAURIN, J. L. MARTIN, R. J. HEADS, C. FOLEY, J. W. MOCKRIDGE, M. J. WRIGHT, Y. WANG, and M. S. MARBER The role of differential activation of p38-mitogen-activated protein kinase in preconditioned ventricular myocytes FASEB J, November 1, 2000; 14(14): 2237 - 2246. [Abstract] [Full Text]
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 M. S. Marber Ischemic Preconditioning in Isolated Cells Circ. Res., May 12, 2000; 86(9): 926 - 931. [Full Text] [PDF]
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 E. Kevelaitis, A. Oubenaissa, J. Peynet, C. Mouas, and P. Menasche Preconditioning by Mitochondrial ATP-Sensitive Potassium Channel Openers : An Effective Approach for Improving the Preservation of Heart Transplants Circulation, November 9, 1999; 100 (2009): II-345 - II-350. [Abstract] [Full Text] [PDF]
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E. Kevelaitis, J. Peynet, C. Mouas, J.-M. Launay, and P. Menasche Opening of Potassium Channels : The Common Cardioprotective Link Between Preconditioning and Natural Hibernation? Circulation, June 15, 1999; 99(23): 3079 - 3085. [Abstract] [Full Text] [PDF]
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 H. Han, H. Wang, H. Long, S. Nattel, and Z. Wang Oxidative Preconditioning and Apoptosis in L-cells. ROLES OF PROTEIN KINASE B AND MITOGEN-ACTIVATED PROTEIN KINASES J. Biol. Chem., July 6, 2001; 276(28): 26357 - 26364. [Abstract] [Full Text] [PDF]
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