Phospholipase D Plays a Role in Ischemic Preconditioning in Rabbit Heart
Background Activation of protein kinase C (PKC) is thought to be a critical step in ischemic preconditioning. Many receptor agonists activate PKC via stimulation of phospholipase C (PLC), which degrades membrane phospholipids to diacylglycerol (DAG), an important PKC cofactor. However, adenosine receptors, critical components of the prototypical preconditioning pathway, are not thought to couple to PLC in the cardiomyocyte. We therefore tested whether ischemic preconditioning or adenosine might instead activate phospholipase D (PLD) to produce DAG.
Methods and Results PLD activity was measured in isolated rabbit hearts. Ischemic injury was evaluated in either isolated rabbit hearts or dispersed myocytes. PLD activity doubled from a control level of 74.8±10.0 to 140.0±11.5 μmol·min−1·g−1 (P<.025) after two 5-minute periods of global ischemia separated by 5 minutes of reperfusion. A similar increase was noted after the heart had been exposed to (R)-N6-(2-phenylisopropyl)-adenosine [(R)-PIA] for 20 minutes. When sodium oleate, which activates PLD, was administered to isolated hearts before a 30-minute coronary occlusion, infarct size (15.6±2.0% of the risk zone) was significantly smaller than in untreated hearts (30.4±2.2%; P<.01). Exposure to sodium oleate significantly prolonged the rate of isolated myocyte survival during simulated ischemia. Propranolol 100 μmol/L, which blocks DAG production from metabolites produced by PLD catalysis, completely abolished the protective effects of both metabolic preconditioning and (R)-PIA exposure in myocytes.
Conclusions We conclude that PLD stimulation is involved in the protection of ischemic preconditioning in the rabbit heart.
Preconditioning is a phenomenon whereby the heart naturally adapts itself to become resistant to infarction during ischemia.1 We have found that preconditioning may also be triggered by agonists to a variety of PKC-coupled cardiac receptors,2 3 4 5 including adenosine A1 receptors.6 In fact, in the rabbit heart, adenosine is believed to be the principal endogenous agonist released by ischemic myocardial cells and responsible for the cardioprotective changes after brief episodes of ischemia. The role of PKC in preconditioning has been confirmed in both rat7 and rabbit8 hearts and isolated rabbit9 and human10 ventricular myocytes. Yet the actual coupling mechanism between adenosine and PKC remains unknown.
α1-Agonists,2 angiotensin II,3 bradykinin,5 and endothelin-1,4 each of which can mimic ischemic preconditioning, are known to activate PKC via PLC.11 12 13 14 When PLC is activated by G protein complexes formed after agonist-receptor interaction, it catalyzes hydrolysis of membrane inositol-containing phospholipids: phosphatidylinositol, phosphatidylinositol 4-monophosphate, and PIP2. PIP2 is hydrolyzed to DAG and IP3 (Fig 1⇓). IP3 causes release of Ca2+ from the sarcoplasmic reticulum, which in turn is believed to result in a positive inotropic action. DAG stimulates the translocation and activation of PKC. The production of inositol phosphates by PLC has become the standard test to determine whether a receptor activates the PLC pathway.
The increase in phosphatidylinositol breakdown products after adenosine treatment of cardiac tissue is too modest to classify adenosine A1 receptors as PLC coupled.15 One attractive explanation is that adenosine receptors may couple to PLD rather than PLC. PLD is another phospholipase that is responsible for degradation of other membrane phospholipids, including phosphatidylcholine, the most prevalent membrane phospholipid. The immediate degradation products are choline and PA; the latter is metabolized further by a phosphohydrolase to DAG. Because IP3 is not formed, one would not expect increased inotropy.
The present study determined whether ischemia or exogenous adenosine could activate PLD, which was monitored with an assay based on a transphosphatidylation reaction uniquely catalyzed by PLD.16 17 We also tested whether an agent that activates PLD could mimic ischemic preconditioning both in rabbit myocytes and in the intact heart. Finally, we inhibited production of DAG from PLD and tested whether that intervention could block the protective effect of preconditioning in rabbit myocytes.
New Zealand White rabbits (weight, 2 to 3 kg) were anesthetized with an intravenous injection of sodium pentobarbital (30 mg/kg), intubated through a tracheotomy, and ventilated with 100% oxygen by a positive-pressure respirator (MD Instruments). A left thoracotomy was performed in the fourth intercostal space. In animals scheduled to be used for in vitro heart studies, a silk suture was passed around a prominent branch of the left coronary artery visible on the epicardial surface. The ends of the suture were passed through a short length of silicone elastomer tubing to form a snare. Tightening of the snare resulted in coronary occlusion as evidenced by the appearance of distal cyanosis and bulging of the noncontracting myocardium, whereas visible hyperemia on the epicardial surface and resumption of myocardial contraction were apparent after loosening of the snare. Hearts were quickly excised and suspended by the aortic root from a Langendorff apparatus.
In Vitro Studies
Hearts were perfused at a pressure of 75 mm Hg with modified Krebs-Henseleit buffer containing (in mmol/L) NaCl 118.5, NaHCO3 24.8, KH2PO4 1.2, CaCl2 2.5, KCl 4.7, MgSO4 1.2, and glucose 10. The perfusate was warmed to 37°C and aerated with 95% O2/5% CO2. After a stabilization period of at least 15 minutes, a recirculating perfusion system was established such that effluent dripping from the heart was collected, passed through a 0.5-μm filter, and pumped into a reservoir, which then resupplied perfusate to the heart. [1-14C]Butanol 250 μCi (3.95 mCi/mmol) diluted in ethanol was then added to the perfusate to produce a final concentration of 20 μmol/L butanol and 0.03% ethanol; the perfusate was allowed to recirculate for 20 minutes. The heart was then quick-frozen with Wollenberger tongs chilled in liquid nitrogen and was removed from the perfusion apparatus. Hearts were maintained at −75°C until assayed for 14C-phosphatidylbutanol, the product of the transphosphatidylation reaction catalyzed by PLD.
Assay for phosphatidylbutanol
14C-Phosphatidylbutanol was extracted, separated, and assayed according to a previously published report.17 Briefly, the heart was powdered and solubilized in a chloroform/methanol mixture. Compounds were separated by TLC on silica gel K6 plates with the organic phase of 2,2,4-trimethylpentane/ethyl acetate/acetic acid/H2O (6:11:2:9 vol/vol/vol/vol) used as solvent. The phosphatidylbutanol band was identified by cochromatography of authentic standard. The isolated band was then scraped into a tube, and radioactivity was measured in a β-scintillation counter.
Rabbits were randomly assigned to five different groups (Fig 2⇓). All hearts experienced 20 minutes of exposure to [1-14C]butanol. In the control group, no interventions were undertaken during the 20-minute period of recirculating [1-14C]butanol. In group 2, we induced global myocardial ischemia for the last 5 minutes of the 20-minute period by arresting the flow of perfusate. The third group (ischemia and reperfusion) experienced 5 minutes of normal perfusion and 5 minutes of global ischemia followed by 10 minutes of reperfusion. Group 4 (the preconditioned group) experienced 5 minutes of normal perfusion, 5 minutes of ischemia, 5 minutes of reperfusion, and then 5 minutes of ischemia. Finally, in group 5, (R)-PIA was added to the perfusate to a final concentration of 200 μmol/L, and the perfusate was allowed to recirculate for 20 minutes. In group 5, there was no episode of ischemia.
To investigate the effect of PLD stimulation, sodium oleate, a putative activator of PLD,17 18 was added to the perfusate. Because sodium oleate could be solubilized only in a solution that contained albumin, BSA was added to the Krebs-Henseleit buffer described above to a final concentration of 0.1%. After stabilization in control hearts, the coronary artery was occluded for 30 minutes. Thereafter, the snare was loosened to allow the heart to reperfuse for 2 hours. In the experimental hearts, 20 μmol/L sodium oleate was added to the perfusate. Hearts were exposed to sodium oleate for 20 minutes starting 15 minutes before coronary occlusion. After the 30-minute occlusion period, the hearts were reperfused for 2 hours.
Measurement of Risk Zone and Infarct Size
After completion of the 2-hour reperfusion period, we demarcated the risk zone by reoccluding the coronary artery with the snare and infusing 1 to 2 mL of a 1% suspension of zinc cadmium sulfide fluorescent microspheres (Duke Scientific Corp) into the perfusate. Thus, the risk zone consisted of nonfluorescing myocardium. The heart was then removed from the perfusion apparatus, frozen, and cut into 2-mm-thick slices. Each slice was stained in 1% triphenyltetrazolium chloride at 37°C for 20 minutes. The slices were then alternately illuminated with ultraviolet and white light, and outlines of the nonfluorescent and infarcted regions were traced on a plastic overlay. Areas of risk zone and infarct were measured by planimetry, and we calculated volumes by multiplying areas by the 2-mm thickness. Infarct was determined as a percent of the risk zone.
Isolated Myocyte Studies
To further test the role of PLD in preconditioning, the isolated myocyte preparation was used. As previously detailed,9 19 isolated rabbit hearts were perfused with buffer containing (in mmol/L) NaCl 125, KCl 30, NaHCO3 25, KH2PO4 1.2, MgCl2 1.2, glucose 11, and l-glutamine 0.68; BSA 1 mg/mL; and a complete amino acid solution (pH 7.4). Ventricular myocytes were isolated by the addition of collagenase to the calcium-free perfusate and maceration of the heart. Viable myocytes were separated by use of slow-speed centrifugation in buffer containing 0.1% BSA. Cells were made calcium tolerant by the slow restoration of calcium in the medium to 1.25 mmol/L. Cells were divided into four to eight tubes for each study. Preparations were considered satisfactory only if rods accounted for >80% of the counted cells at the beginning of each experiment. Ischemia was simulated by centrifugation of myocytes into a pellet (≈0.5 mL of packed cells), and the supernatant was replaced with 0.5 mL of mineral oil to exclude oxygen. Every 30 minutes for 2 hours, a 10-μL aliquot of cells was removed to determine viability/fragility via observation of the ability of the cells to exclude trypan blue dye in a hypotonic (85 mOsm) medium. Those cells unable to exclude the dye were considered dead. Three hundred cells from each tube were counted, and the percentage of stained (dead) cells was determined. A plot of percent of dead cells versus time was constructed. An index of nonsurvival was calculated as the area under the curve after 2 hours and presented as percent/hour. It should be noted that this assay does not strictly measure cell death as the end point but rather the appearance of membrane fragility, which occurs during ischemia and can be delayed by preconditioning.9
Myocytes were preconditioned by incubation in glucose-free medium for 10 minutes, after which glucose was restored for 30 minutes before simulated ischemia was initiated.9 The effect of the PLD activator sodium oleate was determined by the addition of 0, 20, 40, or 80 μmol/L to nonpreconditioned myocytes from the same heart. Cells were exposed to oleate for 10 minutes before simulated ischemia was initiated. For each experiment, suitable controls always included oxygenated cells that were not experiencing simulated ischemia with and without the addition of 80 μmol/L sodium oleate.
Propranolol at a high dose of 100 μmol/L is an effective antagonist of PA phosphohydrolase,17 20 21 the enzyme responsible for converting PA to DAG. Pilot studies revealed that intact rabbit hearts would not tolerate this concentration of propranolol; therefore, we could test it only in the myocyte system. To determine the effect of this agent on preconditioning, myocytes were exposed to the drug starting 10 minutes before the time of preconditioning and continuing through the pelleting phase. In an additional experiment, (R)-PIA 100 μmol/L, an adenosine analogue known to precondition the myocardium,16 was added to the medium for 10 minutes before the cells were centrifuged into a pellet. Finally, the effect of coadministration of (R)-PIA and propranolol was evaluated.
Biochemical Measurement of Phosphatidylbutanol
To measure the direct effect of oleate on PLD activity, the transphosphatidylation reaction was monitored again but this time in the isolated cardiomyocytes by use of a biochemical assay for phosphatidylbutanol. Myocytes were exposed to either 80 or 160 μmol/L sodium oleate and 10 mmol/L n-butanol.22 Cells were incubated in the butanol/propranolol mixture for 10 minutes at 37°C in the presence of the sodium oleate. After the 10-minute exposure to the agonist, lipids were extracted by a modification of the method of Bligh and Dyer.23 Briefly, the reaction was stopped by addition of an ice-cold mixture of methanol/chloroform (2:1) and vortexing the cell suspension. The suspension was then homogenized with a Polytron (Brinkmann Instruments), and suspension remnants were washed from the Polytron with 1 mL chloroform. One milliliter of 200 mmol/L NaCl was added to bring the concentration to 50 mmol/L NaCl in the aqueous phase, and the mixture was centrifuged for 5 minutes at 3000 rpm. The lower lipid phase was separated and the aqueous phase again washed with 1.5 mL chloroform to extract the remaining lipid. After centrifugation at 3000 rpm for 5 minutes, this lipid phase was separated and combined with the first separated lipid phase. The combined lipid fraction was dried under a nitrogen stream and redissolved in 50 μL chloroform.
To separate the lipids, modifications of previously published methods were used.17 K6-TLC plates (Whatman) were activated for 30 to 60 minutes at 120°C. After cooling, plates were spotted with lipid and placed in a TLC chamber with the organic phase of a previously agitated mixture of ethyl acetate:iso-octane:acetic acid:water (10:5:2:5 vol/vol/vol/vol). The plate was run for 60 minutes, after which it was dried and dipped into a solution of 10% CuSO4 and 10% H3PO4.24 The plate was baked for 10 minutes at 160°C to 180°C. Migration of phospholipids in the sample was compared with that of known phosphatidylbutanol and PA samples (Rf=0.11 for PA and Rf=0.24 for phosphatidylbutanol). Known amounts of each lipid standard were spotted on the plates. Phospholipids that migrated to the same level as phosphatidylbutanol and PA were measured by computer-assisted light densitometry.22 24 Protein content of each myocyte sample was assayed with a Bio-Rad kit on 20-μL aliquots of myocytes taken just before addition of butanol. PLD activity was expressed as nanograms of phosphatidylbutanol formed per 10 minutes per milligram of protein.
[1-14C]Butanol was obtained from New England Nuclear. Sodium oleate, BSA, and propranolol were purchased from Sigma Chemical Co. (R)-PIA was obtained from Research Biochemicals, Inc. Collagenase was obtained from Worthington Biochemical Corp.
Results are presented as mean±SEM. Student's unpaired t test and ANOVA with Scheffe´'s test for post hoc testing were used to test for statistical significance. A value of P<.05 was considered to be significant. For the myocyte studies, ANOVA with repeated measures was used.
In Vitro Studies
Each of the five groups consisted of six rabbits except group 2, which had only three hearts. Coronary flow and left ventricular developed pressure were identical among groups and were unchanged throughout the protocol except during the episodes of global ischemia, when flow and pressure declined to zero. However, reperfusion was always accompanied by restoration of normal function. Switching to the butanol-containing perfusate had no effect on mechanical function.
Results of the PLD activity determinations for the five rabbit groups are presented in Fig 3⇓. Baseline activity in the control hearts of group 1 averaged 74.8±10.0 μmol·min−1·g−1. Although activity increased by 46% in group 2 after 5 minutes of ischemia, this trend was not significant. The small number of animals in group 2 may have contributed to this result, but even in group 3, in which ischemia was followed by reperfusion, the 61% increase in PLD activity over control did not quite attain statistical significance. However, the ischemic preconditioning protocol of group 4 significantly increased PLD activity by 87% to 140.0±11.5 μmol·min−1·g−1 (P<.025). Twenty minutes of exposure of the heart to (R)-PIA also significantly increased PLD activity to 132.3±11.1 μmol·min−1·g−1 (P<.05).
Sodium Oleate in Isolated Hearts
Four control and five treated rabbits were studied. Sodium oleate had no effect on either left ventricular developed pressure (120.0±4.5 mm Hg before versus 111.5±1.9 mm Hg after drug) or coronary flow (75.0±4.0 to 72.6±1.3 mL/min). Furthermore, there were no differences between control and experimental groups before coronary occlusion. After reperfusion, there was a tendency for both developed pressure and coronary flow to be higher in oleate-treated hearts, and this difference reached statistical significance after 60 minutes (≈45% versus 65% recovery in control and treated hearts, respectively). As can be seen in Fig 4⇓, hearts treated with sodium oleate had significantly smaller infarcts than untreated hearts (15.6±2.0% versus 30.4±2.2% of the risk zone; P<.01).
Sodium Oleate and Propranolol in Isolated Myocytes
To further explore the influence of PLD on preconditioning, the effects of both PLD stimulation and interference with PLD-generated DAG were evaluated in myocytes free from most influences of endothelial and other cells. Each protocol was repeated six times with cells from different hearts. Fig 5⇓ demonstrates the effect of various concentrations of sodium oleate on cell survival. The figure depicts the progressive cell death in control cells subjected to simulated ischemia without preconditioning. A dose-dependent salvage of cells was observed in the myocytes exposed to oleate for only 10 minutes before simulated ischemia. When the areas under the death curves for the first 120 minutes were compared, the difference between the control (31.4±1.9%·hour) and 80 μmol/L sodium oleate groups (20.3±1.5%·hour) revealed significant protection (P<.05).
Conversely, high-dose propranolol, which by itself had little effect on survival of oxygenated or ischemic cells, completely reversed the salvage of cells observed after preconditioning (Fig 6⇓). The area under the curve for preconditioned cells averaged 34.6±1.4%·hour, which was significantly less than that in control cells subjected to simulated ischemia without preconditioning (45.8±1.6%·hour; P<.05). The latter death curve was virtually identical to that seen in both nonpreconditioned and preconditioned cells exposed to propranolol (45.0±2.4%·hour and 45.3±1.6%·hour, respectively). Therefore, high-dose propranolol did block the protection afforded by preconditioning.
(R)-PIA 100 μmol/L protected myocytes equally as well as preconditioning with glucose deficient medium (31.4±2.2%·hour; P<.05 versus control) (Fig 7⇓). This protection was also abolished by cell exposure to propranolol (43.6±2.8%·hour).
To document that oleate did indeed stimulate PLD, the amount of phosphatidylbutanol produced by myocytes in the presence of butanol was measured by thin-layer chromatography. In unstimulated cells from four hearts, PLD activity averaged 6.0±3.2 ng/mg protein. In the presence of 80 μmol/L sodium oleate, PLD activity in cells from the same four preparations more than doubled to 12.7±4.4 ng/mg protein (P<.05). When isolated myocytes from an additional five hearts were exposed to a doubled concentration of oleate (160 μmol/L), there was no further enhancement of PLD activity (11.2±1.6 ng/mg protein versus 5.3±1.2 ng/mg protein for control hearts; P<.01).
These studies strongly support a role for PLD in ischemic preconditioning. PLD modifies the polar portion of phosphatidylcholine and/or other phospholipids by catalyzing hydrolytic formation of PA with concomitant release of the nonphosphorylated base. PLD also catalyzes the transphosphatidylation reaction in which the base moiety of substrate phospholipid is exchanged with free alcohols (Fig 1⇑). The latter reaction is unique to PLD.25 Randall et al16 greatly simplified monitoring of this reaction by using radioactive butanol as the nucleophilic acceptor, and we used a modification of their technique to evaluate activity of this enzyme. Moraru and colleagues17 previously demonstrated that in vitro assays of PLD in subcellular fractions correlated well with PLD activity assessed by the transphosphatidylation reaction.
In the intact rabbit heart, we found that ischemia increased PLD activity, although the increase reached statistical significance only after two 5-minute periods of global ischemia, a preconditioning protocol. We also found that (R)-PIA, an A1-selective adenosine analogue, increased PLD activity by a similar amount. Thus, exogenous and very likely endogenously released adenosine are capable of stimulating PLD in the rabbit heart. That effect does not appear to be limited to the rabbit heart, because Moraru et al17 also demonstrated that ischemia in the rat heart activates PLD.
Agonists to other PKC-coupled receptors, specifically norepinephrine and endothelin-1, can also stimulate PLD in rabbit ventricular myocytes.22 Carbachol, an agonist of M2-muscarinic receptors, which are thought to couple to the same G proteins as adenosine A1 receptors and can mimic ischemic preconditioning,26 has been reported to stimulate PLD in chicken heart.27
Sodium oleate is reported to be a stimulant of PLD.17 18 We now have observed that oleate can more than double PLD activity in isolated rabbit myocytes as well. In the rat, administration of this fatty acid improves postischemic functional recovery and diminishes lactate dehydrogenase, and creatine phosphokinase release.17 Our studies broaden these observations and demonstrate significantly smaller infarcts in rabbit hearts pretreated with oleate and preserved membrane integrity in isolated ischemic myocytes.
High-dose propranolol is an effective antagonist of PA phosphohydrolase.17 20 21 Accordingly, propranolol prevents production of DAG by PLD and thus blocks the ability of PLD to activate PKC. Propranolol 100 μmol/L caused standstill of the isolated rabbit heart. Consequently, we could evaluate the action of propranolol in the myocyte model only. This dose of propranolol, however, completely reversed the salutary effect of preconditioning by substrate removal or exposure to an adenosine analogue in the isolated myocyte model of simulated ischemia. However, propranolol had no effect on viability in untreated cells. These data strongly implicate PLD and its alternate pathway of phospholipid metabolism in the protection of both ischemic and adenosine-induced preconditioning in the rabbit heart. Different batches of myocytes die at different rates during simulated ischemia. To eliminate this variability as a methodological problem, each batch of myocytes was divided into four groups and studied in parallel. We have found that myocytes from a single batch always die at the same rate.
The present study did not evaluate the importance of the PLC pathways. Many of the agonists to PKC-coupled receptors such as norepinephrine are known to liberate IP3 when administered and therefore use the PLC pathway.11 However, as noted above, many of these agonists also stimulate PLD.22 The PLD-initiated metabolic sequence may be an alternate pathway for intracellular signaling. Because several preconditioning mimetics, such as adenosine, bradykinin, and norepinephrine, are released by the heart during ischemia, we have proposed5 that the pathway to the protection afforded by preconditioning is quite redundant, perhaps representing a built-in safety factor. It appears that this redundancy exists at the phospholipase level as well.
The PLD pathway may be more than just an alternative pathway for the preconditioning phenomenon. In rabbit hearts, protection persists for ≈1 hour after a single 5-minute period of ischemia.28 We originally proposed a translocation theory to explain this “memory,”29 but PLD could also explain it. Phosphatidylinositol degradation by PLC yields a relatively small amount of DAG for a short period of time, peaking at 30 seconds, compared with phosphatidylcholine hydrolysis, which causes a quantitatively larger DAG production and lasts considerably longer.20 This prolonged DAG production by PLD is associated with prolonged translocation of PKC20 and therefore may account for the memory of ischemic preconditioning.
A role for PKC activation in preconditioning remains controversial in pig30 and dog31 hearts. The reason for this discrepancy is unclear at this time; however, there is good evidence that PKC activation is required to precondition the human heart.10 32 The critical role of PKC has also been demonstrated in the preconditioned rat heart.7 33
In conclusion, these studies reveal that PLD plays a role in preconditioning, at least in the rabbit heart. We also found that adenosine A1 receptors can activate this enzyme and that the protective effect of adenosine on rabbit myocytes is dependent on PLD activity. In addition to furthering our understanding of the signal transduction pathways involved in ischemic preconditioning, our findings with PLD reveal yet another potential route for pharmacological preconditioning of the heart.
Selected Abbreviations and Acronyms
|PKC||=||protein kinase C|
This work was supported in part by grants from the National Institutes of Health, National Heart, Lung, and Blood Institute (HL-20648, HL-34360, and HL-50688).
- Received January 22, 1996.
- Revision received April 12, 1996.
- Accepted April 15, 1996.
- Copyright © 1996 by American Heart Association
Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation. 1986;74:1124-1136.
Tsuchida A, Liu Y, Liu GS, Cohen MV, Downey JM. α1-Adrenergic agonists precondition rabbit ischemic myocardium independent of adenosine by direct activation of protein kinase C. Circ Res. 1994;75:576-585.
Goto M, Liu Y, Yang X-M, Ardell JL, Cohen MV, Downey JM. Role of bradykinin in protection of ischemic preconditioning in rabbit hearts. Circ Res. 1995;77:611-621.
Liu GS, Thornton J, Van Winkle DM, Stanley AWH, Olsson RA, Downey JM. Protection against infarction afforded by preconditioning is mediated by A1 adenosine receptors in rabbit heart. Circulation. 1991;84:350-356.
Speechly-Dick ME, Mocanu MM, Yellon DM. Protein kinase C: its role in ischemic preconditioning in the rat. Circ Res. 1994;75:586-590.
Ytrehus K, Liu Y, Downey JM. Preconditioning protects ischemic rabbit heart by protein kinase C activation. Am J Physiol. 1994;266:H1145-H1152.
Armstrong S, Downey JM, Ganote CE. Preconditioning of isolated rabbit cardiomyocytes: induction by metabolic stress and blockade by the adenosine antagonist SPT and calphostin C, a protein kinase C inhibitor. Cardiovasc Res. 1994;28:72-77.
Ikonomidis JS, Shirai T, Weisel RD, Derylo B, Rao V, Whiteside CI, Mickle DAG, Li R-K. ‘Ischemic’ or adenosine preconditioning of human ventricular cardiomyocytes is protein kinase C dependent. Circulation. 1995;92(suppl I):I-12. Abstract.
Kaku T, Lakatta E, Filburn C. α-Adrenergic regulation of phosphoinositide metabolism and protein kinase C in isolated cardiac myocytes. Am J Physiol. 1991;260:C635-C642.
Sadoshima J-i, Izumo S. Signal transduction pathways of angiotensin II-induced c-fos gene expression in cardiac myocytes in vitro: roles of phospholipid-derived second messengers. Circ Res. 1993;73:424-438.
Irons CE, Murray SF, Glembotski CC. Identification of the receptor subtype responsible for endothelin-mediated protein kinase C activation and atrial natriuretic factor secretion from atrial myocytes. J Biol Chem. 1993;268:23417-23421.
Minshall RD, Nakamura F, Becker RP, Rabito SF. Characterization of bradykinin B2 receptors in adult myocardium and neonatal rat cardiomyocytes. Circ Res. 1995;76:773-780.
Billah MM, Anthes JC. The regulation and cellular functions of phosphatidylcholine hydrolysis. Biochem J. 1990;269:281-291.
Fu T, Okano Y, Nozawa Y. Differential pathways (phospholipase C and phospholipase D) of bradykinin-induced biphasic 1,2-diacylglycerol formation in non-transformed and K-ras-transformed NIH-3T3 fibroblasts: involvement of intracellular Ca2+ oscillations in phosphatidylcholine breakdown. Biochem J. 1992;283:347-354.
Ye H, Wolf RA, Kurz T, Corr PB. Phosphatidic acid increases in response to noradrenaline and enodothelin-1 in adult rabbit ventricular myocytes. Cardiovasc Res. 1994;28:1828-1834.
Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959;37:911-917.
Van Winkle DM, Thornton JD, Downey DM, Downey JM. The natural history of preconditioning: cardioprotection depends on duration of transient ischemia and time to subsequent ischemia. Coron Artery Dis. 1991;2:613-619.
Vogt A, Barancik M, Weihrauch D, Arras M, Podzuweit T, Schaper W. Activation of protein kinase C fails to protect ischemic porcine myocardium from infarction in vivo. J Mol Cell Cardiol. 1994;26:CXVIII. Abstract.
Przyklenk K, Sussman MA, Simkhovich BZ, Kloner RA. Does ischemic preconditioning trigger translocation of protein kinase C in the canine model? Circulation. 1995;92:1546-1557.
Speechly-Dick ME, Grover GJ, Yellon DM. Does ischemic preconditioning in the human involve protein kinase C and the ATP-dependent K+ channel? Studies of contractile function after simulated ischemia in an atrial in vitro model. Circ Res. 1995;77:1030-1035.
Mitchell MB, Meng X, Ao L, Brown JM, Harken AH, Banerjee A. Preconditioning of isolated rat heart is mediated by protein kinase C. Circ Res. 1995;76:73-81.