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Circulation. 1996;93:781-791

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(Circulation. 1996;93:781-791.)
© 1996 American Heart Association, Inc.

Articles

Role of Activation of Protein Kinase C in the Infarct SizeLimiting Effect of Ischemic Preconditioning Through Activation of Ecto-5'-nucleotidase

Masafumi Kitakaze, MD; Koichi Node, MD; Tetsuo Minamino, MD; Kazuo Komamura, MD; Hiroharu Funaya, MD; Yoshiro Shinozaki, BS; Mitsuaki Chujo, BS; Hidezo Mori, MD; Michitoshi Inoue, MD; Masatsugu Hori, MD; Takenobu Kamada, MD

From The First Department of Medicine, Osaka (Japan) University School of Medicine; Tokai University School of Medicine (Y.S., M.C., H.M.), Department of Physiology, Isehara (Japan); and Department of Information Science (M.I.), Osaka (Japan) University Hospital.

Correspondence to Masafumi Kitakaze, MD, PhD, The First Department of Medicine, Osaka University School of Medicine, 2-2 Yamadaoka, Suita 565, Japan.


*    Abstract
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 *Abstract
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Background We have reported previously that ischemic preconditioning limits infarct size by increasing ecto-5'-nucleotidase activity. Since we have also reported that protein kinase C activation increases ecto-5'-nucleotidase activity in rat cardiomyocytes, we tested whether activation of protein kinase C during ischemic preconditioning contributes to the infarct size–limiting effect through augmentation of ecto-5'-nucleotidase activity in the canine heart.

Methods and Results The coronary artery was occluded four times for 5 minutes with alternating 5-minute periods of reperfusion (ischemic preconditioning). Then the coronary artery was occluded for 90 minutes followed by 6 hours of reperfusion. Infarct size, normalized by the risk area, in the ischemic preconditioning group was smaller than in the control group (42.6±3.6% in the control group versus 7.9±1.8% in the ischemic preconditioning group, P<.001). Myocardial ecto-5'-nucleotidase activity was increased after the ischemic preconditioning procedure but the increase in ecto-5'-nucleotidase was attenuated by inhibitors of protein kinase C (polymyxin B and GF109203X). Both polymyxin B and GF109203X blunted the infarct size–limiting effect of ischemic preconditioning (infarct size 33.1±6.9% and 35.1±6.4%, respectively). The infarct size–limiting effect was also blunted by an inhibitor of ecto-5'-nucleotidase. Transient administration of methoxamine mimicked the increase in ecto-5'-nucleotidase activity and the infarct size–limiting effect, both of which were abolished by inhibitors of protein kinase C.

Conclusions We conclude that activation of ecto-5'-nucleotidase and protein kinase C contributes to the infarct size–limiting effect of ischemic preconditioning.


Key Words: ecto-5'-nucleotidase • myocardial infarction • adenosine • receptors, adrenergic, alpha • ischemia


*    Introduction
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Brief periods of ischemia that precede sustained ischemia limit infarct size markedly, a phenomenon known as ischemic preconditioning,1 2 3 and the mechanisms underlying this phenomenon have been studied extensively.4 5 6 Recently, Liu et al7 demonstrated that exposure to 8-sulfophenyltheophylline blunts the infarct size–limiting effect of IP in the rabbit heart, and Liu et al7 and Thornton et al8 showed that adenosine A1 receptor activation is responsible for limiting infarct size. Other investigators9 10 11 reached the same conclusion. Furthermore, we reported previously12 13 14 that IP increases ecto-5'-nucleotidase activity, which seems consistent with the results of Liu et al,7 because adenosine production in ischemic myocardium is attributable to ecto-5'-nucleotidase.15 16 On the other hand, {alpha}1-adrenoceptor activation has been shown to mimic the infarct size–limiting effect,17 18 19 which has been confirmed by several investigators. Although these two mechanisms of IP, activation of 5'-nucleotidase and {alpha}1-adrenoceptors, seem to be independent, they appear to be tightly linked, since we have reported20 21 22 that {alpha}1-adrenoceptor activation increases 5'-nucleotidase activity through activation of PKC. Although several laboratories, including ours, reported that activation of PKC mediates IP,23 24 25 26 it has not been demonstrated clearly whether activation of PKC mediates the infarct size–limiting effect of IP through activation of ecto-5'-nucleotidase.

To test this idea, we measured myocardial ecto-5'-nucleotidase activity and infarct size in control and preconditioned myocardium with and without administration of PKC inhibitors. Furthermore, we tested whether IP can activate PKC. Finally, we also tested whether activation of ecto-5'-nucleotidase and the infarct size–limiting effect caused by intermittent exposure to methoxamine are abolished by administration of inhibitors of PKC.


*    Methods
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Instrumentation
Mongrel dogs (weight, 12 to 25 kg) were anesthetized with sodium pentobarbital (30 mg/kg IV), intubated, and ventilated with room air mixed with oxygen (100% O2 at 1.0 L/min). The chest was opened through the left fifth intercostal space, and the heart was suspended in a pericardial cradle. After administration of heparin (500 U/kg IV), we cannulated and perfused the LAD coronary artery with blood from the left carotid artery through an extracorporeal bypass tube. CBF in the perfused area was measured with an electromagnetic flow probe attached to the bypass tube, and CPP was monitored at the tip of the coronary artery cannula. Arterial blood was sampled for blood gas analysis at 60- to 90-minute intervals to monitor the condition of the dogs. The left atrium was catheterized for microsphere injection. Hydration was maintained by a slow normal saline infusion. Heart rate averaged 138±1 bpm during control conditions and did not change during the study. The pH, PO2, and PCO2 in the systemic arterial blood before the protocols were instituted were 7.40±0.02, 107±4 mm Hg, and 38.5±1.9 mm Hg, respectively.

Experimental Protocols
Protocol 1: Effects of Inhibitors of PKC on IP
In the open chest dogs, both CPP and CBF were measured continuously. After hemodynamic stabilization, four cycles of 5 minutes of coronary occlusion and a subsequent 5 minutes of reperfusion were performed to precondition the myocardium to sustained ischemia (IP group; n=8). As a control, instead of IP, the coronary artery was occluded for 90 minutes after 45 minutes of hemodynamic stabilization and was reperfused for 6 hours (control group; n=8).

In seven dogs, constant infusion of polymyxin B (300 µg·kg-1·min-1, 18 mg/mL with an infusion rate of 0.0167 mL·kg-1·min-1) into the LAD coronary artery was performed 5 minutes before and during IP (the polymyxin B with IP group). Polymyxin B (Sigma Chemical Co), an inhibitor of PKC,27 was diluted with saline. In six dogs, polymyxin B was infused into the LAD coronary artery for 45 minutes before ischemia without IP (the polymyxin B group). Since polymyxin B also blocks K+ channels, we also used GF109203X (Calbiochem), a very specific inhibitor of PKC,28 with (the GF109203X with IP group; n=7) and without (the GF109203X group; n=7) IP. Constant infusion of GF109203X (300 µg·kg-1·min-1, 18 mg/mL, with an infusion rate of 0.0167 mL·kg-1·min-1) into the LAD coronary artery was performed 5 minutes before and during IP (the GF109203X with IP group) and for 45 minutes before ischemia without IP (the GF109203X group).

Protocol 2: Effects of Inhibitors of PKC on Methoxamine Exposure–Induced Infarct Size–Limiting Effect
We have reported previously14 that intermittent exposure to methoxamine mimics the infarct size–limiting effect of IP through activation of ecto-5'-nucleotidase activity. To test whether activation of PKC is responsible for this cardioprotection, we administered methoxamine dissolved in saline (40 µg·kg-1·min-1 IC, 2.4 mg/mL, with an infusion rate of 0.0167 mL·kg-1·min-1; Sigma) for four cycles of 5 minutes with 5-minute intervals with and without administration of either polymyxin B or GF109203X (n=6 each for the methoxamine group, the methoxamine with polymyxin B group, and the methoxamine with GF109203X group). After methoxamine exposure with and without administration of either polymyxin B or GF109203X, periods of 90 minutes of coronary occlusion and 6 hours of reperfusion were imposed.

Protocol 3: Effect of IP on Activity of Myocardial PKC
We used 10 other dogs in this protocol. With (n=5; the IP group) and without (n=5; the control group) IP, we measured PKC activity of cytosolic and membrane fractions in the endocardial and epicardial myocardium before sustained ischemia. We quickly sampled myocardial tissue at the conclusion of the protocol and stored it at -80°C.

Protocol 4: Effect of Inhibitors of PKC on Myocardial Ecto-5'-nucleotidase and Cytosolic 5'-Nucleotidase Activity With and Without Either IP or Methoxamine Exposure
We used five dogs in nine groups each: the control group, the IP group, the polymyxin B group, the polymyxin B with IP group, the GF109203X group, the GF109203X with IP group, the methoxamine group, the methoxamine with polymyxin B group, and the methoxamine with GF109203X group. In the control group, we sampled the endocardial and epicardial myocardium after 45 minutes without any interventions. In the polymyxin B group and the GF109203X group, we sampled the endocardial and epicardial myocardium with administration of polymyxin B or GF109203X for 45 minutes. Furthermore, we also examined ecto-5'-nucleotidase activity in the endocardial and epicardial myocardium with IP in the presence of either polymyxin B (300 µg·kg-1·min-1 IC, 18 mg/mL, with an infusion rate of 0.0167 mL·kg-1·min-1) or GF109203X (40 µg·kg-1·min-1 IC, 2.4 mg/mL, with an infusion rate of 0.0167 mL·kg-1·min-1). Finally, we administered methoxamine dissolved in saline (40 µg·kg-1·min-1 IC, 2.4 mg/mL, with an infusion rate of 0.0167 mL·kg-1·min-1) for four cycles of 5 minutes with 5-minute intervals with and without administration of either polymyxin B or GF109203X (the methoxamine group, the methoxamine with polymyxin B group, and the methoxamine with GF109203X group). We used the samples of the control and IP groups in protocol 3 as samples of these groups for protocol 4. We examined 5'-nucleotidase activity in the endocardial and epicardial myocardium. We quickly sampled myocardial tissue at the conclusion of the protocol and stored it at -80°C.

Protocol 5: Effect of Inhibitors of Ecto-5'-nucleotidase on Myocardial Ecto-5'-nucleotidase Activity and the Infarct Size–Limiting Effect of IP
We have previously reported13 that an inhibition of ecto-5'-nucleotidase below the baseline level blunts the infarct size–limiting effect of IP. To test whether prevention of the increases in ecto-5'-nucleotidase activity to the baseline level still blunts the infarct size–limiting effect of IP, we measured ecto-5'-nucleotidase activity of the membrane fraction of the preconditioned myocardium obtained in protocol 4 during various exposures to AMP-CP (0.05, 0.1, 0.5, 1, and 5x10-5 mol/L). Since we found that 0.5x10-5 mol/L AMP-CP (Sigma) reduces the increased ecto-5'-nucleotidase activity to the baseline level, we tested whether the corresponding dose of AMP-CP (8 µg·kg-1·min-1 IC) blunts the infarct size–limiting effect of IP. In six dogs, AMP-CP (8 µg·kg-1·min-1, 0.48 mg/mL, with an infusion rate of 0.0167 mL·kg-1·min-1) was administered into the LAD coronary artery 5 minutes before the IP procedure and was continued for 60 minutes of reperfusion, except for the control occlusion period (the AMP-CP with IP group). In six dogs, AMP-CP was administered into the LAD coronary artery for 40 minutes before ischemia and was continued for 60 minutes of reperfusion, except for the coronary occlusion period (the AMP-CP group).

Criteria for Exclusion
To ensure that all of the animals included in the data analysis of infarct size were healthy and exposed to similar extents of ischemia, the following standards were used to exclude unsatisfactory dogs: (1) subendocardial collateral flow >15 mL/100 g per minute, (2) heart rate >170 bpm, and (3) more than two consecutive attempts required to convert ventricular fibrillation with low-energy DC pulses applied directly to the heart.

Measurement of Infarct Size
After 6 hours of reperfusion in protocols 1 and 2, while the LAD coronary artery was reoccluded and perfused with autologous blood, Evans blue dye was injected into a systemic vein to determine the anatomic risk area and the nonischemic area in the heart. The heart was then removed immediately and sliced into serial transverse sections 6 to 7 mm in width. The nonischemic area was identified by blue stain, and the ischemic region was incubated at 37°C for 20 to 30 minutes with 1% TTC (Sigma) with 0.1 mol/L phosphate buffer adjusted to pH 7.4. TTC stained the noninfarcted myocardium to a brick-red color, indicating the presence of a formazan precipitate caused by reduction of TTC by dehydrogenase enzymes in viable tissues. Infarct size was expressed as a percentage of the area at risk.

For randomization of the study, the area of necrosis and the risk area were measured in all of the dogs at completion of the protocol without knowledge of treatment of each heart.

Measurement of Regional Myocardial Blood Flow
Regional myocardial blood flow was determined by the microsphere technique, which uses nonradioactive microspheres (Sekisui Plastic Co, Ltd) made of inert plastic labeled with different stable heavy elements, as described in detail previously.29 In the present study, microspheres labeled with Br or Zr were used. The mean diameter was 15 µm, and the specific gravity was 1.34 for Br and 1.36 for Zr. Microspheres were suspended in isotonic saline with 0.01% Tween 80 to prevent aggregation. The microspheres were ultrasonicated for 5 minutes followed by 5 minutes of vortexing immediately before injection. Approximately 1 mL of the microsphere suspension (2 to 4x106 spheres) was injected into the left atrium, followed by several warm (37°C) saline flushes (5 mL). Microspheres were administered 80 minutes after the onset of coronary occlusion. Just before microsphere administration, a reference blood flow sample was drawn from the femoral artery at a constant rate of 8 mL/min for 2 minutes.

The x-ray fluorescence of the stable heavy elements was measured by a wave-length dispersive spectrometer (model PW 1480, Phillips Co, Ltd). The specifications of this x-ray fluorescence spectrometer have been described in detail.29 Briefly, when the microspheres are irradiated by a primary x-ray beam, the electrons fall back to a lower orbit and emit measurable energy with a characteristic x-ray fluorescence energy level for each element. Therefore, it is possible to qualify the x-ray fluorescence of several species of labeled microspheres in a single mixture. Myocardial blood flow was calculated according to the formula: time flow=tissue countsx(reference flow/reference counts), and was expressed in mL/100 g per minute. We measured the wet weight of the sampled myocardium.

Measurement of 5'-Nucleotidase and PKC Activities
A biopsy specimen (1 to 2 g) of the myocardium supplied by the LAD coronary artery was obtained before sustained coronary occlusion with and without IP in protocol 3. This specimen was subdivided into endocardial and epicardial halves, and the myocardial tissue samples (0.5 to 1 g each) were frozen and stored under liquid nitrogen. We measured the ecto-5'-nucleotidase and cytosolic 5'-nucleotidase activities in both samples.30

The myocardium was separated into membrane and cytosolic fractions by use of the following technique: Myocardial tissue was homogenized with a Potter-Elvehjem homogenizer (30 strokes) for 5 minutes in 10 vol of ice-cold 10 mmol/L HEPES-potassium hydroxide (HEPES-KOH) buffer (pH 7.4) containing 0.25 mol/L sucrose, 1 mmol/L MgCl2, and 1 mmol/L mercaptoethanol at 0°C. The crude homogenate was strained through a double-layered nylon sieve and homogenized again for 1 minute. For the preparation of membrane and cytosolic fractions, the homogenate was centrifuged at 1000g for 10 minutes, and the supernatant was centrifuged at 200 000g for 1 hour. After this procedure, we regarded the pellet and supernatant fractions as the membrane and cytosolic fractions, respectively. The membrane and cytosolic fractions were dialyzed at 4°C for 4 hours against 10 mmol/L HEPES-KOH (pH 7.4) containing 1 mmol/L MgCl2, 1 mmol/L mercaptoethanol, and 0.01% activated charcoal and were divided into aliquots that were frozen immediately and stored at -80°C.

5'-Nucleotidase activity was assessed by the enzymatic assay technique30 and was reported as nanomoles per milligram of protein per minute. Protein concentration was measured by the method of Lowry et al31 with bovine serum albumin used as a standard. 5'-Nucleotidase activity of membrane and cytosolic fractions was defined as ecto-5'-nucleotidase and cytosolic 5'-nucleotidase activity, respectively. When cytosolic 5'-nucleotidase activity was measured, AMP-CP (50 µmol/L) was added to prevent contamination of ecto-5'-nucleotidase. In a preliminary study, we examined the recovery of 5'-nucleotidase activity in the membrane fraction with use of this procedure and found that the recovery of ecto-5'-nucleotidase was 97±2% (n=5). This recovery rate is highly reproducible.12 13

The activity of PKC was measured by enzyme assay with the RPN 77A kit (Amersham), which provides a simple and reliable method of estimating PKC without extensive purification of the samples.22 Activity of PKC was expressed as nanomoles per milligram of protein per minute. Protein concentrations were measured by the method of Lowry et al31 with bovine serum albumin used as a standard. Furthermore, to examine the Ca2+ and phospholipid dependency of PKC activity, we measured PKC activity by adding 0.5 mmol/L in excess of EGTA to chelate Ca2+ and eliminated phosphatidyl-L-serine from the assay system.22

Statistical Analysis
Statistical analyses were performed with paired and unpaired t tests,32 33 and the significance level was adjusted according to a modified Bonferroni correction. To compare data among groups, a modified Bonferroni correction was used to determine significance (P<.05) for group pairs that exhibited statistically significant differences.32 33 ANCOVA, with regional collateral flow in the inner-half left ventricular wall as the covariant, was used to account for the effect of collateral blood flow on infarct size. Each value was expressed as mean±SEM, with a value of P<.05 considered significant.


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Mortality and Exclusions
One hundred and seven dogs were randomly assigned to 11 groups for assessment of infarct size (Table 1Down). Fifteen dogs were excluded from data analysis because subendocardial collateral flow was >15 mL/100 g per minute. Therefore, 92 dogs completed the protocol satisfactorily and were used for data analysis. Of the 92 dogs, 26 developed ventricular fibrillation at least once. Among these 26 dogs, ventricular fibrillation that matched the exclusion criteria occurred in 7 dogs during 90 minutes of ischemia and in 12 dogs during reperfusion after 90 minutes of ischemia. These animals were excluded from assessment of infarct size.


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  		Table 1. Number of Dogs Assigned and Excluded in Each Group

Changes in Hemodynamic Parameters and Myocardial 5'-Nucleotidase Activity During IP and Intermittent Exposure to Methoxamine
Systolic and diastolic blood pressures and heart rate before, during, and after 90 minutes of myocardial ischemia were compared in the nine groups (Table 2Down). There were no significant differences in systolic and diastolic blood pressures and heart rate among the nine groups. Before and after IP and during 40 minutes of hemodynamic stabilization, neither CPP nor CBF (105±5 mm Hg and 91±2 mL/100 g per minute, respectively, at baseline in the control group) changed significantly in any of the groups. In the IP group, coronary hyperemic flow during reperfusion after brief periods of ischemia was observed (90±2 to 332±16 mL/100 g per minute, P<.001). Administration of neither polymyxin B nor GF109203X during IP procedure changed the extent of reactive hyperemic flow during reperfusion. CBF decreased after administration of methoxamine in the methoxamine group (92±2 to 74±3 mL/100 g per minute, P<.01) but returned to the control level 5 minutes after the fourth exposure to methoxamine (88±2 mL/100 g per minute). CPP increased during methoxamine administration (106±5 to 133±6 mm Hg, P<.01) but returned to the control level 5 minutes after the fourth exposure to methoxamine (102±5 mm Hg). Neither polymyxin B nor GF109203X affected both CPP and CBF during methoxamine administration.


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  		Table 2. Sequential Changes in Systolic and Diastolic Blood Pressure Before, During, and After 90 Minutes of Ischemia

IP significantly increased both ecto-5'-nucleotidase activity (Fig 1Down) and cytosolic 5'-nucleotidase activity (Fig 2Down) in the myocardium. Administration of polymyxin B and GF109203X blunted the increases in ecto-5'-nucleotidase and cytosolic 5'-nucleotidase activity caused by IP. Methoxamine increased both ecto-5'-nucleotidase and cytosolic 5'-nucleotidase activity to the levels obtained with IP, which was also blunted by polymyxin B and GF109203X administrations. AMP-CP (0.05, 0.1, 0.5, 1, and 5x10-5 mol/L) reduced the increased ecto-5'-nucleotidase activity from 74.8±2.2 to 67.9±4.2, 59.0±1.4, 41.3±4.5, 22.9±3.4, and 5.0±1.7 nmol/mg protein per minute (n=5, P<.001).



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  		Figure 1. Comparison of canine ecto-5'-nucleotidase activity in the epicardium (A) and endocardium (B) after 45 minutes of steady state observation (control group, n=5), IP (n=5), IP with GF109203X administration (n=5) and with polymyxin B administration (n=5), and intermittent exposure to methoxamine (MTX) with GF109203X administration (n=5) and polymyxin B administration (n=5) and without GF109203X and polymyxin B (n=5). IP increased ecto-5'-nucleotidase activity, which was blunted by inhibitors of PKC and mimicked by MTX.



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  		Figure 2. Comparison of canine cytosolic 5'-nucleotidase activity in the epicardium (A) and endocardium (B) after 45 minutes of steady state observation (control group, n=5), IP (n=5), IP with GF109203X administration (n=5) and with polymyxin B administration (n=5), and intermittent exposure to methoxamine (MTX) with GF109203X administration (n=5) and polymyxin B administration (n=5) and without GF109203X and polymyxin B (n=5). IP increased ecto-5'-nucleotidase activity, which was blunted by inhibitors of PKC and mimicked by MTX.

Activity of PKC in Control Myocardium and Preconditioned Myocardium
To investigate whether IP increases the PKC activity of the myocardium, we measured PKC activity of the membrane and cytosolic fractions of the control myocardium and preconditioned myocardium. Figs 3Down and 4Down show PKC activity of the membrane and cytosolic fractions in the epicardial and endocardial myocardium, respectively. PKC activity in the membrane fraction was increased in the preconditioned myocardium. In contrast, PKC activity of the cytosolic fraction did not increase in the IP group. Depletion of Ca2+ and/or phospholipids decreased the basal activity of PKC of the cytosolic but not of the membrane fraction. On the other hand, depletion of Ca2+ and/or phospholipids blunted activation of PKC of the membrane but not of the cytosolic fraction. Thus, the increase in PKC activity of the membrane fraction in preconditioned myocardium was dependent both on Ca2+ and phospholipids.



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  		Figure 3. PKC activity in the membrane and cytosolic fractions in control and preconditioned epicardium. Activity of PKC was measured in the presence (+) and absence (–) of Ca2+ and/or phospholipids (PL).



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  		Figure 4. PKC activity in the membrane and cytosolic fractions in control and preconditioned endocardium. Activity of PKC was measured in the presence (+) and absence (–) of Ca2+ and/or phospholipids (PL).

The Infarct Size–Limiting Effect of IP and Its Relation to PKC Activity
Ninety minutes of coronary occlusion did not change systemic hemodynamic parameters in any of the groups (Table 2Up). Fig 5Down shows the risk area and collateral flow in all groups. The risk area and collateral flow were comparable in all of the groups. Fig 6Down shows infarct size in each of the groups. IP markedly attenuated infarct size, and GF109203X, polymyxin B, and AMP-CP completely abolished the infarct size–limiting effect of IP. With methoxamine administration, infarct size was attenuated to the level seen with IP (Fig 6Down). However, the infarct size–limiting effect caused by exposure to methoxamine was blunted by GF109203X and polymyxin B administration. GF109203X, polymyxin B, and AMP-CP themselves did not affect infarct size caused by 90 minutes of myocardial ischemia and subsequent reperfusion. Fig 7Down illustrates the regression plots of infarct size as a percentage of the area at risk against collateral flow in the preconditioned and control groups, with and without either AMP-CP, polymyxin B, or GF109203X administration. IP significantly and markedly reduced infarct size. AMP-CP, polymyxin B, and GF109203X each blunted the infarct size–limiting effect at every level of collateral flow. Furthermore, transient methoxamine administration mimicked the infarct size–limiting effect of IP, which was also blunted by polymyxin B and GF109203X.



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  		Figure 5. Risk area and collateral flow during ischemia in the control group (n=8), the IP group (n=8), the GF109203X with IP group (n=8), the GF109203X group (n=7), the polymyxin B with IP group (n=7), the polymyxin B group (n=6), the methoxamine (MTX) group (n=6), the MTX with GF109203X group (n=6), and the MTX with polymyxin B group (n=6). There were no differences in the risk area and collateral flow during ischemia in any of the groups. In the figure, AOPCP indicates AMP-CP.



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  		Figure 6. Infarct size in the control group (n=8), the IP group (n=8), the GF109203X with IP group (n=7), the GF109203X group (n=7), the polymyxin B with IP group (n=7), the polymyxin B group (n=6), the methoxamine (MTX) group (n=6), the MTX with GF109203X group (n=6), and the MTX with polymyxin B group (n=7). Infarct size was markedly decreased in the IP group. The infarct size–limiting effect of IP was completely abolished by administration of GF109203X and polymyxin B. On the other hand, MTX mimicked the infarct size–limiting effect of IP, which was blunted by GF109203X and polymyxin B. In the figure, AOPCP indicates AMP-CP.



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  		Figure 7. Plot of infarct size expressed as a percentage of the risk area and regional collateral flow during ischemia. In the control group (n=8), the IP group (n=8), the GF109203X with IP group (n=7), the GF109203X group (n=7), the polymyxin B with IP group (n=7), the polymyxin B group (n=6), the methoxamine (MTX) group (n=6), the MTX with GF109203X group (n=6), and the MTX with polymyxin B group (n=6), there are inverse relations between normalized infarct area and collateral flow. Infarct size in the preconditioned hearts vs control hearts was smaller (P<.001) with respect to any given value for collateral blood flow. Infarct size was markedly increased (P<.001) in the GF109203X and polymyxin B with IP groups compared with the IP group. Infarct size in the GF109203X with IP group, the GF109203X group, the polymyxin B with IP group, and the polymyxin B group was not different. On the other hand, transient MTX exposure mimicked the infarct size–limiting effect of IP, which was blunted by GF109203X and polymyxin B. Statistical significance was tested by ANCOVA. In the figure, AOPCP indicates AMP-CP.


*    Discussion
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Linkage Between the Infarct Size–Limiting Effect and Activation of Ecto-5'-nucleotidase Through PKC Activation in IP
IP has been the focus of intense study by basic and clinical investigators, because a number of laboratories have confirmed that IP markedly limits infarct size.1 2 3 12 13 14 15 16 17 18 19 20 However, subcellular mechanisms must be elucidated if IP is to be applied to the treatment of acute myocardial infarction. In the present study, we have reported that cardioprotection due to IP is attributable to activation of PKC and subsequent activation of ecto-5'-nucleotidase.

In the present study, we observed that IP translocates PKC from cytosolic fractions to membrane fractions, indicating that IP activates PKC in the canine myocardium. This observation is consistent with the observation of Strasser et al.34 However, there is a report35 that PKC is not activated 10 minutes after the IP procedure. Since we measured PKC activity 5 minutes after the IP procedure, PKC translocated to the membrane fraction returned to the cytosolic fraction in several minutes. This indicates that activation and translocation of PKC caused by IP is transient. Indeed, persistent activation of PKC is reported to adversely expand infarct size.36 This suggests that persistent activation of PKC during ischemia and reperfusion is not essential, but phosphorylation of certain proteins by PKC before sustained ischemia may be required for cardioprotection caused by IP. Since we have revealed that activated PKC is Ca2+ and phospholipid sensitive, activation of conventional PKC may be involved.

There are several ways to activate PKC during IP. First, since adenosine is reported to trigger the infarct size–limiting effect of IP in the rabbit heart, adenosine may activate PKC through G proteins. Indeed, Strasser et al37 reported that endogenous adenosine can activate PKC during ischemia. In the canine heart, we showed38 that transient exposures to high doses of adenosine can activate ecto-5'-nucleotidase and limit infarct size; however, we also observed38 that administration of 8-sulfophenyltheophylline during the IP procedure blunts neither the infarct size–limiting effect nor activation of ecto-5'-nucleotidase in canine hearts. This result suggests that endogenous adenosine during the IP procedure may not reach the level required to trigger the cardioprotection that is possibly caused by activation of PKC. In rat cardiomyocytes, exposure to adenosine adversely decreased ecto-5'-nucleotidase activity,39 which corresponds with the observation that cardioprotection caused by IP is not attributable to adenosine in rat hearts. We previously reported14 that endogenous norepinephrine during the IP procedure is responsible for activation of ecto-5'-nucleotidase and mediates the infarct size–limiting effect, because prazosin blunted the infarct size–limiting effect and activation of ecto-5'-nucleotidase. Furthermore, we also reported22 that {alpha}1-adrenoceptor activation increases ecto-5'-nucleotidase activity in rat cardiomyocytes through activation of PKC. These observations indicate that endogenous norepinephrine40 can trigger the infarct size–limiting effect of IP in the canine heart. This observation has been confirmed by several investigators. In the rabbit heart, Tsuchida et al19 reported that phenoxybenzamine administration during the IP procedure cannot blunt the infarct size–limiting effect of IP, although phenylephrine can trigger the infarct size–limiting effect. In the rabbit heart, recent study41 indicates that bradykinin is involved in the infarct size–limiting effect of IP through activation of PKC. The contribution of bradykinin to cardioprotection41 may be large in the rabbit and may cause differences in the infarct size–limiting effect of IP in rabbit and dog models.

In rabbit hearts, Tsuchida et al19 reported that methoxamine does not cause the infarct size–limiting effect of IP, but phenylephrine does, suggesting that activation of {alpha}1b-adrenoceptors is involved in cardioprotection. In contrast, we showed that methoxamine mimics the infarct size–limiting effect of IP.14 Tsuchida et al19 argue against the dose of methoxamine we use; they suspect that the dose of methoxamine used in the present study is large and may stimulate not only {alpha}1a-adrenoceptors but also {alpha}1b-adrenoceptors. However, even low doses of methoxamine (10-9 to 10-5 mol/L) can activate ecto-5'-nucleotidase,22 which suggests that {alpha}1a-adrenoceptor stimulation can activate ecto-5'-nucleotidase through activation of PKC. Indeed, activation of {alpha}1a-adrenoceptors activates PKC, although stimulation of {alpha}1b-adrenoceptors does not.42 43 To establish which receptors are responsible for cardioprotection, we need to further investigate differences in species using more specific antagonists and agonists. Furthermore, Endoh et al44 did not find the coupling of the {alpha}-adrenoceptor activation–inositol 1,4,5-triphosphate accumulation–myocardial contraction in canine and rabbit myocardia. The present and previous studies20 39 from our laboratory suggest linkage of the {alpha}1-adrenoceptor activation–PKC–ecto-5'-nucleotidase activation in the canine heart. One possibility to explain the difference between our studies and the work of Endoh et al44 is that linkage of {alpha}-adrenoceptor activation–IP3 accumulation–myocardial contraction is not complete, but linkage of {alpha}1-adrenoceptor activation–PKC–ecto-5'-nucleotidase activation may exist in the canine myocardium.

Although there may be differences in species concerning how PKC is activated during IP, many investigators, including our group, agree that activation of PKC is involved in cardioprotection caused by IP. Indeed, Ytrehus et al23 showed that activation of PKC mimics the infarct size–limiting effect. As for the upstream mechanism of cardioprotection caused by activation of PKC, we have suggested that activation of ecto-5'-nucleotidase may be responsible for such cardioprotection. In the present study, activation of ecto-5'-nucleotidase and the infarct size–limiting effect are abolished by administration of inhibitors of PKC, and methoxamine-induced cardioprotection is also blunted by administration of inhibitors of ecto-5'-nucleotidase. Furthermore, 8 µg·kg-1·min-1 AMP-CP IC, which blunts the increases in ecto-5'-nucleotidase activity to the baseline level, blunted the infarct size–limiting effect. These results strongly support the idea that activation of PKC increases ecto-5'-nucleotidase activity and mediates the infarct size–limiting effect. We have also reported that activation of ecto-5'-nucleotidase due to PMA and norepinephrine results in the acquisition of cardioprotective ability against hypoxia and reoxygenation in rat cardiomyocytes, and this cardioprotection is also blunted by concomitant administration of AMP-CP.45 These results suggest that activation of ecto-5'-nucleotidase caused by activated PKC is a primary mediator for the infarct size–limiting effect of IP. Since cytosolic 5'-nucleotidase was also activated by the IP procedure in the present study and activation of PKC did not increase cytosolic 5'-nucleotidase activity in rat cardiomyocytes, all of the effects of the infarct size–limiting effect of IP may not be attributable to the linkage of activation of PKC and ecto-5'-nucleotidase.

Although we have not elucidated the mechanisms whereby PKC increases ecto-5'-nucleotidase activity, we speculate that PKC may change the characteristics of the active site of ecto-5'-nucleotidase or induce a conformational change in the structure of ecto-5'-nucleotidase.

Linkage Between Activation of Ecto-5'-nucleotidase and the Infarct Size–Limiting Effect of IP
Since we showed that ecto-5'-nucleotidase is activated by PKC, these data may indicate that adenosine release is increased during ischemia in the IP group. However, there are several reports that IP causes slower degradation of adenine nucleotides and less production of purine nucleosides, including adenosine, in the ischemic myocardium.1 4 5 46 These observations seem contradictory to ours, but they are not. First, since we did not measure adenosine concentration of myocardial tissue samples, we did not have direct evidence to support or deny these observations. Second, levels of myocardial adenosine and adenine nucleotides are independent of extracellular adenine nucleotides and adenosine levels, because extracellular adenosine concentration depends on the activity of ecto-5'-nucleotidase and the concentration of 5'-AMP, and intracellular adenine nucleotide levels depend on the energy state of cardiomyocytes. We also indirectly observed intracellular adenosine production during ischemia and reperfusion using AMP-CP13 : AMP-CP reduced adenosine release to the level of the control group with reduction of ecto-5'-nucleotidase activity to one tenth of the baseline control value,13 indicating that the increased release of adenosine in response to ischemia and reperfusion is attributable to the activation of ecto-5'-nucleotidase. This conclusion is consistent with the work of Imai et al.15 Furthermore, extracellular AMP, a substrate for ecto-5'-nucleotidase, is reported to exist in the intracellular space to produce adenosine.15 47 Therefore, even if levels of adenosine and adenine nucleotides in the myocardial tissue are low during sustained ischemia, extracellular adenosine levels near the plasma membrane can be higher in the preconditioning group. When adenosine production in the cellular surface is high, adenosine A1-receptor–mediated energy-sparing effects may preserve high-energy phosphates and cause less degradation of high-energy phosphates and adenine nucleotides in cardiomyocytes.

There is a report that increases in adenosine concentration in the interstitial space are not augmented during sustained ischemia after IP,48 although adenosine release during reperfusion is augmented in the IP group.12 13 49 When ecto-5'-nucleotidase is activated in the IP group, the concentration of adenosine surrounding ecto-5'-nucleotidase is thought to increase, which may elevate interstitial adenosine levels. One possibility to explain this difference between Van Wylen's results48 and ours is that adenosine uptake into the myocytes may be enhanced during ischemia in the IP group. The second possibility is the involvement of other enzymes responsible for adenosine production. Adenosine concentration is mainly determined by (1) 5'-nucleotidase and (2) activity of the enzymes involved in the degradation or salvage of adenosine, ie, adenosine deaminase and adenosine kinase.16 The involvement of these factors may alter the interstitial concentration of adenosine produced via ecto-5'-nucleotidase. Third, since we measured adenosine in the coronary venous blood, its level was largely affected by endothelial ecto-5'-nucleotidase. In turn, interstitial adenosine levels may be affected by myocardial ecto-5'-nucleotidase. Thus, IP differently activates ecto-5'-nucleotidase located at endothelial cells and cardiomyocytes. Fourth, it is possible that even if the adenosine concentration in the microenvironment surrounding ecto-5'-nucleotidase on the cellular membrane is increased by the activation of ecto-5'-nucleotidase, the alteration of interstitial volume determined by myocardial cellular swelling and the rate of washout due to lymphatic stream may change the interstitial adenosine concentration. Fifth, considering that the diameter of the microdialysis tube is 300 µm50 and the diameter of the cardiomyocytes is {approx}15 µm, the microdialysis tube may cause considerable cellular damage,50 which may elevate interstitial adenosine levels. Furthermore, the microdialysis tube may not be properly located at the interstitial space. Therefore, this technique includes several technical errors for detection of accurate interstitial adenosine levels. In any of these possible situations, temporal and topical increases in the adenosine concentration surrounding ecto-5'-nucleotidase may be able to directly activate the adenosine receptors located at the same cellular membrane, which may not contradict Van Wylen's results.48 This close juxtaposition may explain how 5'-nucleotidase activates the adenosine receptors. Further investigation is necessary to examine this hypothesis concerning the relationship between activation of ecto-5'-nucleotidase activity and adenosine production in IP.


*    Selected Abbreviations and Acronyms
 
AMP-CP = {alpha},ß-methyleneadenosine 5'-diphosphate
bpm = beats per minute
CBF = coronary blood flow
IP = ischemic preconditioning
LAD = left anterior descending
PKC = protein kinase C
TTC = 2,3,5-triphenyltetrazolium chloride


*    Acknowledgments
 
This study was supported by a grant-in-aid for scientific research (No. 03670449) from the Ministry of Education, Science, and Culture, Japan.

Received July 12, 1995; revision received September 15, 1995; accepted September 25, 1995.


*    References
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*References
 

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