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Am J Physiol Heart Circ Physiol 273: H1860-H1866, 1997;
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Vol. 273, Issue 4, H1860-H1866, October 1997

Ischemic preconditioning triggers phospholipase D signaling in rat heart

Arpad Tosaki1, Nilanjana Maulik1, Gerald Cordis1, Ovidiu C. Trifan1, Laurentiu M. Popescu2, and Dipak K. Das1

1 Cardiovascular Division, Department of Surgery, University of Connecticut School of Medicine, Farmington, Connecticut 06030; and 2 Department of Cell Biology, Carol Davila University of Medicine and Pharmacy, Bucharest 2, Romania

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Recent studies have indicated that repeated brief episodes of ischemia and reperfusion render the myocardium more tolerant to subsequent lethal ischemic injury. In view of the previous observations that ischemia-reperfusion potentiates phospholipase D signaling and that such signaling is beneficial for the heart, we investigated whether a similar phospholipase D signaling is responsible for the beneficial effects associated with repeated ischemia and reperfusion. Using an isolated perfused working rat heart model, we demonstrated that four brief episodes of 5 min of ischemia and 10 min of reperfusion reduced the incidence of ventricular arrhythmias, enhanced the postischemic ventricular performance, and decreased the release of creatine kinase from the reperfused heart, with simultaneous activation of phospholipase D generating the second messengers diacylglycerol and phosphatidic acid and leading to the translocation and activation of protein kinase C. The specific antiphospholipase D antibody blocked the activation of phospholipase D and attenuated the generation of diacylglycerol and phosphatidic acid and activation of protein kinase C. In concert, phospholipase D inhibition increased the incidence of ventricular arrhythmias, blocked the beneficial effects of preconditioning on the ventricular performance, and increased the amount of creatine kinase release from the coronary effluent. The results of this study indicate that repeated brief episodes of ischemia and reperfusion exert beneficial effects on the intact rat heart by triggering the activation of a phospholipase D signaling mechanism.

ischemia-reperfusion; signal transduction; mitogen-activated protein kinases; protein kinase C; tyrosine kinase

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

ISCHEMIC PRECONDITIONING provides a powerful anti-ischemic protection for the heart. For example, ischemic preconditioning induced by repeated short-term ischemia and reperfusion has been found to reduce postischemic left ventricular functional abnormalities (13, 16, 18), ventricular arrhythmias (15, 30), infarct size (8, 26), and cell damage (28). Although preconditioning is a powerful tool in protecting the myocardium from ischemic reperfusion injury, considerable debate remains regarding its mechanism of action. Most of the existing studies were focused on the role of adenosine A1 receptors in preconditioning (26). In addition, alpha 1-adrenergic receptors (29), muscarinic receptors (3), ATP-dependent potassium channels (9), multiple receptors including bradykinin and angiotensin II receptors (17), and G protein (27) have been implicated in ischemic preconditioning. Irrespective of the signaling pathways involved, it is more or less universally accepted that the intracellular signaling leads to the translocation and activation of protein kinase C (PKC) (14). Involvement of PKC has been demonstrated directly by biochemical and immunologic studies (20) and indirectly by experiments demonstrating that PKC inhibitors block the beneficial effects of preconditioning (33).

The primary step of the signal transduction pathway for the activation of PKC involves the stimulation of phospholipase C, generating the second messenger diacylglycerol (24). Several recent studies demonstrated that activation of phospholipase D plays a crucial role in ischemic preconditioning (4). Phospholipase D preferentially attacks phosphatidylcholine, generating phosphatidic acid, which is readily metabolized by a phosphohydrolase present in the heart into diacylglycerol. Activation of phospholipase D was documented in the ischemic-reperfused (23) as well as in the preconditioned hearts (4, 32). Agonists of phospholipase D simulated the effects of ischemic preconditioning, whereas the inhibition of this phospholipase blocked the beneficial effects of preconditioning.

The present study was designed to confirm the involvement of phospholipase D in the ischemic preconditioning. Using specific polyclonal antibodies to phospholipase D, we found that these antiphospholipase D antibodies caused direct inhibition of phospholipase D, simultaneously reducing the amount of diacylglycerol and phosphatidic acid as well as significantly inhibiting the stimulation of PKC. In concert, this antiphospholipase D antibody blocked the beneficial effects of ischemic preconditioning as evidenced by the increased incidence of ventricular arrhythmias.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Isolated working rat heart preparation. Sprague-Dawley rats weighing ~300 g were anesthetized with intraperitoneal pentobarbital sodium (60 mg/kg). After heparin sodium (500 IU/kg) was administered to the rats intravenously, the chests were opened, and the hearts were rapidly excised and mounted on a nonrecirculating Langendorff perfusion apparatus (11). Retrograde perfusion was established at a pressure of 100 cmH2O with an oxygenated normothermic Krebs-Henseleit bicarbonate (KHB) buffer with the following ion concentrations (in mM): 118.0 NaCl, 24.0 NaHCO3, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.7 CaCl2, and 10.0 glucose. The KHB buffer had been previously equilibrated with 95% O2-5% CO2, pH 7.4 at 37°C. Hearts were first perfused with KHB buffer for 5 min for washout, followed by perfusion with antiphospholipase D antibody for 30 min. Antiphospholipase D polyclonal antibody raised in rabbits immunized with phospholipase D (from Streptomyces chromofuscus, Sigma Chemical, St. Louis, MO) was previously used in another study (19). Control experiments were performed by perfusing the hearts with nonimmune immunoglobulin G (IgG) for the same time period. After we perfused (recirculating system) with antiphospholipase D antibody or IgG, 250 µCi [1-14C]butanol (New England Nuclear, Boston, MA) were added to the perfusate (final concentration 22 µM), and the perfusion was continued for an additional period of 15 min. The perfusion was then switched to working mode as described previously (7). The protocol of the experiment is depicted in Fig. 1.


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  		Fig. 1.   Schemata of experimental protocol. KHB, Krebs-Henseleit bicarbonate buffer; IgG, immunoglobulin G; PLD, phospholipase D; 4xPC, 4 cycles of preconditioning.

An epicardial electrocardiogram (ECG) was recorded by a polygraph during the experiment using two silver electrodes attached directly to the heart. The ECGs were analyzed to determine the incidence of ventricular fibrillation (VF) and ventricular tachycardia (VT) and whether they were nonsustained (i.e., spontaneously reverting to regular rhythm) or sustained (persisting throughout the reperfusion period). The heart was considered to be in VF if an irregular undulating baseline was apparent on ECG. VT was defined as five or more consecutive premature ventricular complexes, and this classification included ventricular flutter and repetitive monomorphic VT, which was difficult to dissociate from rapid VT. In each case, VT switched spontaneously to sinus rhythm or VF, and hence, VT was considered nonsustained. The heart was considered to be in sinus rhythm if normal sinus complexes occurring in a regular rhythm were apparent on ECG.

To examine the effects of preconditioning on myocardial functions, aortic flow and developed pressure were measured as described previously (19). The aortic flow was monitored using a calibrated rotameter while the developed pressure was determined as the difference between aortic end-systolic and aortic end-diastolic pressure measured through an on-line pressure transducer.

After baseline ECG recordings were performed, hearts were made globally ischemic for 5 min followed by 10 min of reperfusion (1 time preconditioning, 1xPC). This process was repeated four times to induce repeated ischemia and reperfusion (4xPC). All hearts were then subjected to 30-min ischemia followed by 30-min reperfusion.

In a separate group of experiments, isolated rat hearts were preperfused for 30 min in the presence of nonimmune IgG or antiphospholipase D antibody. All hearts were then perfused for 15 min with sodium oleate (20 µM final concentration) added to the KHB buffer. Hearts were then perfused for an additional period of 30 min with KHB buffer only. At the end of the experiments, hearts were frozen in liquid nitrogen for subsequent analyses of phospholipase D, diacylglycerol, phosphatidic acid, and PKC.

Assay for creatine kinase release. Coronary effluents from rat hearts were collected before preconditioning and during the reperfusion (at 10, 20, and 30 min) to measure creatine kinase (CK) release using a CK assay kit obtained from Sigma Chemical.

Estimation of phospholipase D, diacylglycerol, phosphatidic acid, and PKC. After each experiment, the heart was frozen at liquid nitrogen temperature. At a later date, lipids from the frozen heart muscle were extracted as described elsewhere (6). During the extraction of lipids, 0.005% butylated hydroxytoluene, an antioxidant, was added to prevent breakdown of lipids. Lipids were separated by thin-layer chromatography (TLC) using a silica gel G plate (Whatman, Clifton NJ). Diacylglycerol and phosphatidic acid were identified by the cochromatography with authentic standards. Quantification of the lipids was performed with a scanning densitometer.

Phospholipase D was assayed after separating the extracted lipids by TLC on silica gel K6 plates (Whatman) using the organic phase of 2,2,4-trimethylpentane-ethyl acetate-acetic acid-H2O (6:11:2:9, by volume) as solvent system (23). The phosphatidylbutanol band was identified by cochromatography of authentic standard. The bands were scraped off the plates, and radioactivity incorporated was quantitated using a beta -scintillation counter.

For estimation of PKC, enzyme activities were measured in both cytosolic and particulate fractions. These two fractions were prepared according to the previously described methods (31). Briefly, hearts were homogenized in a buffer containing 20 mM tris(hydroxymethyl)aminomethane (Tris) · HCl, 250 mM sucrose, 2 mM ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid, 4.5 mM EDTA, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 250 µg/ml trypsin inhibitor, 1 mM benzamidine, and 0.005% leupeptin, pH 7.4. Homogenates were centrifuged at 14,000 g for 20 min. The resulting supernatants were centrifuged again at 105,000 g for 90 min. The final supernatant was used as cytosolic fraction. Samples were frozen in liquid N2 and stored at -70°C until use. The pellets from 14,000-g centrifugation were rehomogenized in the same buffer and centrifuged at 14,000 g for 20 min. The pellets obtained were discarded. The supernatants were centrifuged again at 105,000 g for 90 min. Supernatants were discarded, and the pellets from the two 105,000-g centrifugations were combined and resuspendend in a homogenizing buffer containing 0.3% Triton X-100 (vol/vol) using a hand-held homogenizer. This fraction is again centrifuged at 105,000 g for 45 min. This final supernatant serves as particulate fraction. The resulting supernatant (solubilized membrane) and the cytosolic fractions were subjected to ion-exchange chromatography using diethylaminoethyl columns (bed volume 2 ml). After application of the samples to the column by quantity, the columns were washed with 10 ml of the above buffer. PKC was eluted with the same buffer but with the inclusion of 400 mM NaCl. PKC activity was determined according to the method described by Hannun et al. (10). Aliquots containing 10-15 µg of total protein were used in the PKC reaction. The reaction mixture contained in a final volume of 100 µl 50 mM Tris · HCl, pH 7.5, 10 mM DTT, 15 mM magnesium acetate, 150 µM ATP, and 1 µCi [gamma -32P]ATP. Reactions were divided and carried out with 4 mM Ca2+, 1 µM phorbol 12-myristate 13-acetate, and 65 µg/ml L-alpha -phosphatidyl-L-serine (PS), PS only, and with 4 mM EDTA but no PS. Epidermal growth factor receptor peptide (250-300 µM) was used in our reactions as substrate. Reactions were carried out at 25°C for 30 min and terminated by adding 100 µl of 75 mM orthophosphoric acid. One hundred fifty microliters of this reaction mixture were spotted onto a P-81 phosphocellulose paper and washed with 75 mM orthophosphoric acid, and radioactivity was counted in a liquid scintillation counter. The specific activity was calculated by subtracting kinase activity in the presence of 4 mM EDTA and no PS from Ca2+ plus PS-stimulated kinase activity.

Statistical analysis. Results are expressed as means ± SE. A one-way analysis of variance was first performed to test any difference between the mean values of different groups. If differences were established, the results between two groups were compared by Dunnett's test. An analog procedure was followed for distribution of discrete variables such as the incidence of VF and VT. An overall chi 2 test for 2 × n table was constructed, followed by a sequence of 2 × 2 chi 2 tests to compare individual groups. Results were considered significant for P < 0.05.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effects of antiphospholipase D antibody on myocardial arrhythmias. The incidence of reperfusion arrhythmias during the brief episodes of preconditioning was zero in each case (Fig. 2). Thus the incidence of total VF (sustained plus nonsustained) and VT was also zero. In the control group, all hearts showed 100% VF upon reperfusion, 92% of which were in sustained VF. There was no significant difference in incidence of VF in the nonpreconditioned control and IgG-treated groups. Four cycles of preconditioning (4xPC) afforded an antiarrhythmic effect as evidenced by the reduction of the incidence of VF and VT from 100 in each case to 42 and 50%, respectively (P < 0.05). Antiphospholipase D antibody (50 µg/ml) increased the incidence of VF and VT to 93%.


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  		Fig. 2.   Effects of antiphospholipase D antibody and ischemic preconditioning on ventricular fibrillation (VF; A) and ventricular tachycardia (VT; B). Hearts were perfused in presence of either IgG or antiphospholipase D antibody before preconditioning. Hearts were then made ischemic for 30 min followed by 30 min of reperfusion. 1, Time-matched control ischemia and reperfusion without preconditioning; 2, same as 3 but treated with IgG before preconditioning; 3, preconditioned; 4, same as 3 but treated with antiphospholipase D antibody before preconditioning. Results are means of 12 experiments/group. * P < 0.05 compared with control.

Effects of antiphospholipase D antibody on postischemic ventricular functions. We also studied the ventricular performances of the preconditioned hearts in the presence and absence of antiphospholipase D antibody. Myocardial functions were measured at the baseline level and at the end of the experiments, i.e., preconditioning followed by 30 min of ischemia and 30 min of reperfusion. As shown in Table 1, heart rate and coronary flow were not affected by preconditioning or by antiphospholipase D antibody treatment. Aortic flow, developed pressure, and its maximum first derivative were significantly improved in the preconditioned group. The beneficial effects of preconditioning were completely abolished for the preconditioned hearts pretreated with antiphospholipase D antibody.

                              
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  		Table 1.   Effect of antiphospholipase D antibody on ventricular function in preconditioned myocardium

Effects of antiphospholipase D antibody on CK release. CK release from the coronary effluent increased steadily and progressively in all groups of postischemic hearts (Fig. 3). There was no difference between control and IgG-treated groups. Preconditioning reduced the CK release for all the time points. Inhibition of phospholipase D by antiphospholipase D antibody increased the amount of CK release to above baseline levels, suggesting increased myocardial injury.


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  		Fig. 3.   Effects of antiphospholipase D antibody and ischemic preconditioning on creatine kinase (CK) release from heart. Hearts were perfused in presence of either IgG or antiphospholipase D antibody before preconditioning. Hearts were then made ischemic for 30 min followed by 30 min of reperfusion. Amount of CK released from heart was estimated using a CK assay kit as described in MATERIALS AND METHODS. black-down-triangle , Antiphospholipase D antibody; black-square, control; bullet , IgG; black-triangle, preconditioning. Results are means of 12 experiments/group. * P < 0.05 compared with control.

Effects of phospholipase D inhibition on signal transduction. Ischemic preconditioning caused the activation of phospholipase D by ~400% (Fig. 4). IgG did not change the enzyme activity. Antiphospholipase D antibody reduced the phospholipase D activity to ~20% of the baseline value.


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  		Fig. 4.   Effects of antiphospholipase D antibody and ischemic preconditioning on phospholipase D activity in heart. Hearts were perfused in presence of either IgG or antiphospholipase D antibody before preconditioning. Hearts were then made ischemic for 30 min followed by 30 min of reperfusion. Amount of phospholipase D activity was assayed as described in MATERIALS AND METHODS. Results are means of 12 experiments/group. * P < 0.05 compared with control.

Diacylglycerol (Fig. 5, top) and phosphatidic acid (Fig. 5, bottom) followed a similar pattern. Ischemic preconditioning stimulated the generation of these two second messengers by 400 and 600%, respectively. Again, inhibition of phospholipase D resulted in the complete inhibition of phosphatidic acid; on the contrary, diacylglycerol was reduced, only partially suggesting that this messenger molecule is also produced by a different pathway.


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  		Fig. 5.   Effects of antiphospholipase D antibody and ischemic preconditioning on diacylglycerol and phosphatidic acid contents of heart. Hearts were perfused in presence of either IgG or antiphospholipase D antibody before preconditioning. Hearts were then made ischemic for 30 min followed by 30 min of reperfusion. Amounts of diacylglycerol and phosphatidic acid were determined as described in MATERIALS AND METHODS. Results are means of 12 experiments/group. * P < 0.05 compared with control. ** P < 0.05 compared with preconditioning.

Ischemia-reperfusion is known to translocate and activate PKC. We, therefore, measured PKC activity both in membrane and cytosolic fractions. As expected, preconditioning resulted in the translocation of PKC from the cytosol to membrane fraction, resulting in an increase in membrane-to-cytosol ratio (Fig. 6). About a sixfold increase in the ratio as compared with that of baseline (or IgG treated) level was observed in the preconditioned myocardium. Antiphospholipase D antibody partially blocked this enhancement of PKC activity.


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  		Fig. 6.   Effects of antiphospholipase D antibody and ischemic preconditioning on protein kinase C activity in heart. Hearts were perfused in presence of either IgG or antiphospholipase D antibody before preconditioning. Hearts were then made ischemic for 30 min followed by 30 min of reperfusion. Protein kinase C was estimated in both membrane and cytosol. Results are expressed as membrane-to-cytosol ratio. Results are means of 12 experiments/group. * P < 0.05 compared with control. ** P < 0.05 compared with preconditioning.

Effects of oleate and antiphospholipase D antibody. As expected, sodium oleate induced the activation of phospholipase D (Table 2). In concert, increased formation of the phospholipase D-catabolized products diacylglycerol and phosphatidic acid were also noticed by the sodium oleate, which also caused the activation of PKC.

                              
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  		Table 2.   Effects of oleate and antiphospholipase D antibody on formation of phosphatidic acid, diacylglycerol, and protein kinase C catalyzed by phospholipase D

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The heart possesses the remarkable ability to adapt itself against any stressful situation by increasing resistance to the adverse consequences. Stress induced by single or multiple brief periods of ischemia and reperfusion renders the heart more tolerant to the subsequent lethal ischemic insult. This phenomenon has been termed ischemic preconditioning and has been shown to be associated with a delay in the development of tissue necrosis, decrease in postischemic contractile dysfunction, reduction in the severity of arrhythmias, and decrease in the infarct size (3, 8, 9, 13-18, 20, 26-30, 33). However, the mechanism of preconditioning remains far from clear.

In this study, we have demonstrated that antiphospholipase D antibody almost completely inhibited the preconditioning-mediated activation of phospholipase D. In concert, preconditioning generated diacylglycerol and phosphatidic acid and led to the translocation and activation of PKC. Additionally, preconditioning reduced the incidence of ventricular arrhythmias, increased the postischemic ventricular recovery, and decreased the tissue necrosis as evidenced by the reduction of CK release. These beneficial effects of preconditioning were reversed by phospholipase D inhibition.

In a previous study, we found that the same antiphospholipase D antibody blocked ischemia-reperfusion-mediated activation of phospholipase D (22). The antiphospholipase D antibody was produced by immunization with phospholipase D purified from S. chromofuscus. In vitro, this antibody bound to phospholipase D and decreased the specific transphosphatidylation reaction (using n-[1-14C]butanol) to ~10% of the baseline values. To examine whether the antiphospholipase D antibody would indeed bind to phospholipase D in the rat heart, isolated rat hearts were perfused for 30 min with antiphospholipase D antibody (control hearts were simultaneously perfused with nonimmune rabbit IgG) followed by 30 min of perfusion with anti-rabbit IgG labeled with 10 nm gold particles (Sigma Chemical) and subsequently prepared for transmission electron microscopy as described previously (25). Examination of the electron micrographs revealed that the antiphospholipase D antibodies (the gold particles) bound to the outer side of the sarcolemma (data not shown). The binding is specific, since no gold particles were seen in the hearts perfused with nonimmune rabbit IgG. In the present study, perfusion of rat heart with antiphospholipase D antibody decreased the specific transphosphatidylation activity of endogenous phospholipase D to ~30% from the baseline level and prevented any rise in phosphatidic acid level after preconditioning. The results of these experiments clearly implicate that the polyclonal antiphospholipase D antibody used in our study bound to the membrane fraction of phospholipase D.

To further confirm the myocardial phospholipase D inhibition by antiphospholipase D antibody, we perfused the hearts with sodium oleate, a known stimulator of phospholipase D. Sodium oleate activated phospholipase D, which was also reflected by the increased formation of phosphatidic acid and diacylglycerol. In concert, PKC was activated. Antiphospholipase D antibody almost completely blocked the increased effects phospholipase D, indicating that the antibody indeed inhibited the phospholipase D in the heart.

Recently, phospholipase D has been found to play a role in myocardial protection afforded by ischemic preconditioning (4, 32). This enzyme catalyzes the hydrolysis of the terminal diester bond of phosphatidylcholine with the formation of choline and phosphatidic acid (12); the latter serves as a substrate for diacylglycerol biosynthesis by the action of phosphatidic acid phosphohydrolase. Diacylglycerol may serve as a second messenger, leading to the activation of PKC. Phospholipase D also catalyzes a transphosphatidylation reaction in which the phosphatidyl moiety of the phospholipid is transferred to a nucleophilic alcohol, producing corresponding phosphatidylalcohol (1). Ischemia-reperfusion was shown to activate phospholipase D, generating intracellular phosphatidic acid, part of which converted to diacylglycerol (23). Additionally, activation of myocardial phospholipase D by sodium oleate resulted in a significant improvement of postischemic functional recovery and attenuation of cellular injury, suggesting that phospholipase D signaling in the ischemic myocardium is beneficial for the recovery of the heart.

In addition to phospholipase D, other phospholipases including phospholipase A2 and phospholipase C also become activated in response to ischemia and reperfusion (25). However, activation of phospholipase A2 causes the generation of arachidonic acid and lysophosphoglycerides, which are detrimental to the heart (5). Activation of phospholipase C also generates diacylglycerol, but it causes the production of inositol 1,4,5-trisphosphate, which mobilizes Ca2+ from the intracellular compartments (21). Thus, unlike the detrimental effects of phospholipase A2 and phospholipase C, activation of phospholipase D is beneficial to the heart. Activation of phospholipase D produces phosphatidic acid, whereas activation of phospholipase C produces diacylglycerol. However, these two products of hydrolysis are easily convertible, catalyzed by two enzymes, diacylglycerol kinase and phosphatidic acid phosphohydrolase. Inhibition of diacylglycerol kinase does not affect the amount of ischemia-reperfusion-induced generation of diacylglycerol or phosphatidic acid; on the contrary, phosphatidic acid phosphohydrolase inhibition leads to an enhancement of phosphatidic acid in concert with a reduction in diacylglycerol (23), suggesting that phospholipase D activity is solely responsible for the generation of phosphatidic acid. Additionally, it indirectly contributes to diacylglycerol generation, since part of the phosphatidic acid is hydrolyzed to diacylglycerol by phosphatidic acid phosphohydrolase.

Ischemic preconditioning or myocardial adaptation to ischemia has been universally accepted as the state-of-the-art technique for myocardial preservation. However, considerable debate exists concerning the intracellular signaling mechanism of preconditioning. Recent studies suggest that activation of alpha 1-receptor and PKC plays a role in ischemic preconditioning (29, 31). Although PKC can be activated by both phospholipase C and phospholipase D pathways, alpha 1-receptor signaling is mediated by the former pathway. A number of previous studies demonstrated phospholipase C-dependent phosphoinositide degradation and turnover in the ischemic reperfused myocardium (24). It is not unlikely that this pathway may become activated by ischemic preconditioning, which represents multiple episodes of ischemia and reperfusion. Nevertheless, ischemic preconditioning has been found to activate phospholipase D in both rat and rabbit hearts (4, 32). A recent study from our laboratory has also demonstrated preconditioning-induced increase in phospholipase D in swine myocardium (unpublished data). Unlike the alpha 1-receptor signaling pathway, which seems to be species specific, the phospholipase D pathway seems to be valid for multiple animal species.

Involvement of phospholipase D in ischemic preconditioning is further supported by our recent findings of tyrosine kinase signaling of phospholipase D-coupled activation of multiple kinases including mitogen-activated protein (MAP) kinases, MAP kinases-activated protein kinase 2, and PKC (19). Phospholipase D-mediated stimulus-response couplings are known to exist in several cell types (2). In our study, ischemic preconditioning-mediated activation of phospholipase D was found to be inhibited by a tyrosine kinase blocker, genistein (19). In concert, the preconditioning effect was almost abolished by the genistein treatment. Additionally, preconditioning of the rat hearts stimulated multiple protein kinases that were inhibited by genistein, suggesting a role of tyrosine kinase-coupled phospholipase D pathway for ischemic preconditioning and implicating the involvement of multiple protein kinases in myocardial adaptation to ischemia.

In summary, using incidence of arrhythmia and CK release as markers for myocardial recovery, we have shown in this study that inhibition of phospholipase D using a specific antibody blocked the beneficial effects of preconditioning, simultaneously attenuating the generation of diacylglycerol and phosphatidic acid and inhibiting the translocation and activation of PKC. These results suggest that ischemic preconditioning triggers the phospholipase D signaling in ischemic myocardium, which appears to be beneficial for heart.

    ACKNOWLEDGEMENTS

This study was supported by National Heart, Lung, and Blood Institute Grants HL-22559, HL-34360, and HL-33889 and by a grant-in-aid from the American Heart Association.

    FOOTNOTES

Address for reprint requests: D. K. Das, Cardiovascular Division, Dept. of Surgery, Univ. of Connecticut, School of Medicine, Farmington, CT 06030-1110.

Received 24 January 1997; accepted in final form 30 June 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Heart Circ Physiol 273(4):H1860-H1866
0363-6135/97 $5.00 Copyright © 1997 the American Physiological Society


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