Journal Logo

Article

Infarct-Size Limitation by Preconditioning is Enhanced by Dipyridamole Administered Before But Not After Preconditioning: Evidence for the Role of Interstitial Adenosine Level During Preconditioning as a Primary Determinant of Cardioprotection

Suzuki, Katsuo; Miura, Tetsuji; Miki, Takayuki; Tsuchida, Akihito; Shimamoto, Kazuaki

Author Information
Journal of Cardiovascular Pharmacology: January 1998 - Volume 31 - Issue 1 - p 1-9
  • Free

Abstract

Preconditioning the heart with a brief episode of ischemia enhances myocardial tolerance in a variety of animal models of infarction (1,2). In most of these models, except for rat models, the contribution of adenosine-receptor activation has been demonstrated: adenosine-receptor blockers prevented cardioprotection by preconditioning, and adenosine-receptor agonists mimicked the preconditioning effect (1,2). Furthermore, a recent study in this laboratory (3) showed that infarct-size limitation by an A1-receptor agonist, R-phenylisopropyladenosine, was completely abolished by protein kinase C inhibitors, which suggests that protein kinase C activity is a mechanism downstream to the A1 receptor in cardioprotection. These findings also support the protein kinase C hypothesis of preconditioning (2,4,5). According to this hypothesis, the primary difference between a preconditioned and nonpreconditioned myocardium is the status of protein kinase C: upregulation of this enzyme in the preconditioned myocytes causes phosphorylation of a protein (or proteins) during ischemic insult, leading to cardioprotection against infarction. However, it is still not clear what determines the extent of cardioprotection by preconditioning.

Earlier studies from this (6) and other laboratories (7,8) showed that the infarct size-limiting effect of preconditioning is correlated with preconditioning ischemia ranging 2-10 min, which suggests that the amount of triggers produced during preconditioning, such as adenosine (2) and bradykinin (9,10), may be influential. In addition, adenosine receptors may need to be activated during sustained ischemia (11,12), although the interstitial adenosine level during this period is significantly reduced by preconditioning (13,14). However, it has not been investigated whether the level of interstitial adenosine during sustained ischemia determines the preconditioning effect. Accordingly, we hypothesized two possible relations between the interstitial adenosine level and the potency of cardioprotection by preconditioning. One possibility is that not only the interstitial adenosine level during preconditioning but also the adenosine level during sustained ischemia determines the cardioprotective effect. If this is indeed the case, any interventions to restore the ischemia-induced increase of interstitial adenosine in the preconditioned myocardium would potentiate the infarct size-limiting effect of preconditioning. An alternative possibility is that a slight increase of the interstitial adenosine level is necessary during sustained ischemia, and thus enhancement of interstitial adenosine accumulation in this period has no significant effect on preconditioning. To test these possibilities, our study assessed the effect of dipyridamole on the interstitial adenosine level and the relation between the effect of dipyridamole on the infarct size-limiting effect of preconditioning, duration of preconditioning ischemia, and the timing of the administration of this nucleoside-transport inhibitor (i.e., before vs. after preconditioning).

METHODS

Experiment 1. Effect of dipyridamole on the cardiac interstitial purines

Surgical preparation. Male rabbits (Japanese White) weighing 2.2-2.7 kg were anesthetized by pentobarbital (30 mg/kg, i.v.), and additional anesthesia was given during the experiment as needed. A tracheotomy was performed, and the animal was mechanically ventilated with a Harvard respirator (model 683, Harvard Apparatus, South Natick, MA, U.S.A.) using room air and oxygen supplement. The tidal volume, respiratory rate, and oxygen supplement were adjusted to maintain arterial blood gas within the physiologic range. A fluid-filled catheter was inserted into the carotid artery and connected to a Nihon-Kohden (Tokyo, Japan) SCK-590 transducer to monitor blood pressure. Another catheter was placed in the jugular vein for infusing drugs, and precordial electrocardiography was monitored by using bipolar leads across the chest. The heart was exposed via a left thoracotomy, and 4-0 silk was passed around a marginal branch of the left circumflex artery with a taper needle. The ends of the silk thread were threaded through a small vinyl tube to make a snare. In vivo microdialysis was performed, as previously reported (15). In brief, a microdialysis probe (Eicom OP-100-05; Eicom Co., Kyoto, Japan), consisting of a 5-mm-long dialysis fiber (220 μm OD; 200 μm ID; molecular weight cutoff, 50,000) glued between two polyethylene (PE) tubes, was inserted by using a 26G needle into the midmyocardium in the territory of the marginal branch of the circumflex. The probe was perfused with Ringer solution (Na, 147 mM; K, 4 mM; Ca, 4.5 mM; and Cl, 155.5 mM) containing low-molecular-weight heparin (10 U/ml) at 2 μl/min. The preparation was stabilized for 2 h before collecting baseline samples.

Experimental protocols. All rabbits received two episodes of 2-min coronary artery occlusion, separated by a 33-min reperfusion interval. After the first episode of ischemia and subsequent 33-min reperfusion, the rabbits were randomly divided into the control and DIP groups. The former received vehicle (saline), and the latter group was given 0.25 mg/kg of dipyridamole (Behringer-Ingelheim, Tokyo, Japan) intravenously. Ten minutes after the intravenous injection, the coronary artery was reoccluded for 2 min and then reperfused for 33 min. The first test episode of ischemia was set up for matching the two experimental groups, which showed a comparable increase in purines, as pilot experiments showed a large variation in purine data on a rabbit-to-rabbit basis, which could mask the difference by dipyridamole treatment. The dialysate samples were collected every 10 min (baseline and preischemic periods) or every 5 min (after coronary occlusion) into Eppendorff vials and stored at −20°C until assay. Sample collection was started after taking into account the dead space in the dialysis probe and the perfusion rate.

Postmortem analysis of the heart. After the last dialysate sample was taken, the rabbit was given an intravenous bolus of 2,000 U heparin and killed by a pentobarbital overdose. The heart was excised, mounted onto a Langendorff apparatus, and perfused with saline. The coronary branch was reoccluded, and Monastral blue was then injected into the perfusion line negatively to mark the ischemic region. The location of the dialysis probe in the myocardium was checked, and if a part or the whole of the dialysis window was found to be in the nonischemic region, exposed in the ventricular lumen, the data were excluded from the following analysis.

Analysis of purines in the dialysate. Purines in the dialysate were analyzed by a high-performance liquid chromatography (HPLC) system, consisting of an EP-300 pump, Eicompack MA-5 ODS column (length, 150 mm; inner diameter, 4.6 mm), ATC-300 Column Oven (Eicom), Soma UV-VIS Detector/S-3702 (Soma-Kogaku Co., Tokyo, Japan), a Signal Cleaner SC-77 (16), and a data processing-recording unit CC-21 (System Instruments Co., Tokyo, Japan). For detecting low levels of adenosine in the dialysate, we used the Signal Cleaner SC-77, which markedly improves the signal-to-noise ratio in chromatograms (16). Adenosine, inosine, and hypoxanthine were separated with a 0.1 M KH2PO4 buffer (pH 3.5) containing 1% acetonitrile at a flow rate of 1 ml/min. Of 20 μl dialysate obtained during each 10-min sampling period, 15 μl was injected into the reverse-phase column, which was maintained at 25°C in the column oven, and the absorbance was measured at 260 nm. As the dialysate volume was insufficient when the sampling period was 5 min, each 10-μl sample was given the same volume of Ringer solution, and then 15 μl of the diluted sample was injected into HPLC. Peaks were identified and quantified by comparison of retention times and peak heights with known external standards. Dialysate nucleoside concentrations were expressed as μM. In this system, the recovery of nucleosides in the in vitro dialysis of the standard solution (containing 1 ng/μl of each nucleoside) at a perfusion rate of 2 μl/min, 37°C, was as follows: 17.9 ± 0.8% for adenosine, 19.4 ± 0.9% for inosine, and 26.9 ± 1.1% for hypoxanthine, as reported previously (15).

Experiment 2. Effect of dipyridamole on infarct size-limiting effect of preconditioning

Surgical preparation. Rabbits were anesthetized and prepared as in Experiment 1, except for the placement of the microdialysis probe. According to Experimental protocol (see the following), the rabbit was subjected to a pretreatment, and the coronary artery was occluded by the snare for 30 min. Myocardial ischemia was confirmed by regional cyanosis and ST-segment change in the electrocardiogram (ECG). After 30-min coronary occlusion, the snare was released, and the ischemic region was reperfused, which was confirmed by reactive hyperemia in the previously cyanotic region. Three hours after reperfusion, 2,000 U of heparin was intravenously injected, and then the rabbit was killed by a pentobarbital overdose. The heart was quickly removed and processed for postmortem analysis.

Postmortem analysis. The heart was mounted on a Langendorff apparatus and perfused with saline to wash out the remaining blood. The coronary branch was reoccluded, and fluorescent particles (3-30 μm in diameter; Duke Scientific Co., Palo Alto, CA, U.S.A.) suspended in saline was infused into the perfusion line negatively to mark the area at risk (i.e., region of the occluded artery). The heart was then frozen and sectioned into 2-mm transverse slices. The heart slices were incubated in 100 mM sodium phosphate buffer (pH = 7.4) containing 1% triphenyltetrazolium for 20 min to visualize infarcts (17). The slices were then mounted on a glass press, which compressed the heart slices into exactly 2 mm in thickness. A clear acetate sheet was laid over the glass press. The infarct (i.e., region unstained by tetrazolium) was traced under room light, and areas at risk were traced under ultraviolet light on the acetate sheet. The traces were enlarged 2 times by using a Xerox copy machine. The areas of the infarct and region at risk were measured by PIAS II, a computer-assisted analysis system (PIAS Co., Osaka, Japan), and each volume was calculated by multiplying the area by slice thickness.

Experimental protocols

The animals were divided into nine groups as shown in Fig. 1. The control group received the 30-min coronary artery occlusion without any pretreatment. The DIP group was given an intravenous injection of 0.25 mg/kg dipyridamole 5 min before 30-min coronary occlusion. In the 2′PC group, the heart was preconditioned with 2-min ischemia followed by 5-min reperfusion before the 30-min coronary occlusion. The DIP-2′PC and 2′PC-DIP groups received dipyridamole (0.25 mg/kg, i.v.) at 5 min before preconditioning and at 3 min after the preconditioning ischemia, respectively. In the 3′PC group, preconditioning was performed with 3-min ischemia followed by 5-min reperfusion. The DIP-3′PC and 3′PC-DIP groups were given the same dose of dipyridamole at 5 min before preconditioning and at 3 min after the preconditioning ischemia, respectively. In the 3′PC-SPT group, 7.5 mg/kg of SPT (Research Biochemical Inc., Natick, MA, U.S.A.) was given immediately after 3 min of preconditioning ischemia. This dose of SPT was selected as one to block infarct limitation by preconditioning with 5-min ischemia and not to modify infarct size in the nonpreconditioned rabbit heart (18). Because our previous studies (6,19,20) showed that preconditioning with 2-min ischemia causes a very modest, statistically insignificant cardioprotection against infarction, we did not examine the effect of SPT administration after 2-min preconditioning in this study.

FIG. 1
FIG. 1:
Experimental protocols in Experiment 2. Shaded zone, coronary occlusion; open zone, normal perfusion and reperfusion; solid arrow, DIP injection (0.25 mg/kg); open arrow, SPT injection (7.5 mg/kg). PC, preconditioning; DIP, dipyridamole; SPT, 8-sulfophenyltheophylline.

Statistical analysis

The differences in the time course of purine levels in the dialysate and hemodynamic parameters between the experimental groups were examined by two-way repeated-measures analysis of variance (ANOVA). The difference in infarct size between the groups was tested by one-way ANOVA. When the F test showed an overall difference, a comparison of the two groups was performed by the Student-Newman-Keuls post hoc test. A p value < 0.05 was considered statistically significant. Results are expressed as means ± SEM.

RESULTS

Experiment 1

Hemodynamic parameters. Seventeen rabbits (10 in the control and seven in the DIP groups) were initially enrolled in this study, and 13 of these rabbits were selected to match adenosine/inosine levels during the first ischemic period between the two study groups. Table 1 summarizes the hemodynamic data in this series of experiments. The heart rate and mean arterial pressure were comparable between the control and the DIP groups in the preocclusion period and during the first coronary occlusion. Administration of dipyridamole slightly reduced the blood pressure, but there was no statistically significant difference in the blood pressure and heart rate before and during the second occlusion of the coronary artery between these two groups.

TABLE 1
TABLE 1:
Hemodynamic data in experiment 1

Dialysate purine levels.Figure 2 shows representative chromatograms of adenine nucleosides in dialysate samples. The baseline levels of nucleosides were not different between the control group (adenosine, 0.07 ± 0.01 μM; inosine, 0.21 ± 0.03 μM; hypoxanthine, 0.79 ± 0.06 μM) and DIP group (adenosine, 0.06 ± 0.02 μM; inosine, 0.26 ± 0.02 μM; hypoxanthine, 0.82 ± 0.10 μM). Alteration of the nucleoside levels in the dialysate by two episodes of ischemia/reperfusion is illustrated in Fig. 3. In the control rabbits, the adenosine level reached 0.18 ± 0.02 μM (p < 0.05 vs. baseline) during 2-min ischemia and then gradually decreased to the baseline level over the 33-min reperfusion period. During the second episode of ischemia, the adenosine level significantly increased again but reached only 0.10 ± 0.02 μM, which was significantly lower than the peak achieved by the first episode of ischemia. After the second ischemia, the adenosine level in the dialysate immediately returned to the baseline value. In the DIP group, there was no significant difference in the increase of the adenosine level between the first and the second episodes of ischemia, whereas the adenosine level during the first episode of ischemia was comparable to that in the control group (Fig. 3A). The time course of dialysate adenosine before and after the second episode of ischemia was significantly different between the control and DIP groups (by two-way repeated-measures ANOVA). This finding suggests that this dose of dipyridamole could augment the increase in interstitial adenosine during myocardial ischemia. A similar pattern of alteration was observed for the inosine and hypoxanthine levels in both of the two study groups (Fig. 3B and C). Although the level of inosine during the second episode of ischemia was slightly lower than that in the DIP group, this difference failed to reach statistical significance.

FIG. 2
FIG. 2:
Representative chromatograms in Experiment 1. A: Chromatogram of a dialysate sampled under baseline condition. B: Chromatogram of a dialysate sampled during 2-min ischemia and 3-min reperfusion period. Baseline sample was undiluted before injection into the HPLC. The sample obtained during ischemia/reperfusion was diluted with the same volume of dialysis buffer, and 15 μl of the diluted sample (i.e., 7.5 μl of the original sample) was injected into the HPLC (see text for details). ADO, adenosine; INO, inosine; HYPOX, hypoxanthine.
FIG. 3
FIG. 3:
Profile of dialysate purines in untreated control and dipyridamole-treated rabbits. A: Dialysate adenosine. B: Dialysate inosine. C: Dialysate hypoxanthine. Saline or dipyridamole was administered 10 min before the second ischemia. Although the peak level of adenosine after the second ischemia was significantly lower than that after the first ischemia in the control group, the level was maintained at a level similar to that after the first ischemia in the DIP group, indicating enhancement of adenosine accumulation by dipyridamole treatment. *p < 0.05 vs. the peak value after the first episode of ischemia. Shaded zones, 2-min periods of ischemia.

Experiment 2

Hemodynamic data. Hemodynamic parameters in Experiment 2 are summarized in Table 2. Under the baseline condition, the heart rate and mean arterial pressure were comparable in all groups. Although the heart rate did not significantly change throughout the experiment in all the study groups, the mean arterial pressure in the dipyridamole-treated groups was slightly lower during ischemia than in the groups without dipyridamole treatment.

TABLE 2
TABLE 2:
Hemodynamic data in experiment 2

Infarct-size data. As shown in Table 3, heart weight and risk-area size were similar among the nine study groups. Clearly to illustrate the effects of dipyridamole on preconditioning, infarct size expressed as a percentage of area at risk is presented in Figs. 4 and 5. As shown in Fig. 4, there was no significant difference in the infarct size as a percentage of the area at risk (%IS/AR) between the control and DIP groups (41.2 ± 4.7% vs. 38.8 ± 5.8%; p = NS), indicating that dipyridamole pretreatment alone does not modify infarct size. Preconditioning reduced infarct size, and the extent of the protective effect was correlated with the duration of preconditioning ischemia: %IS/AR was smaller in the 3′PC group than in the 2′PC group (20.5 ± 1.8% vs. 32.1 ± 4.7%). Furthermore, potentiation of infarct-size limitation by dipyridamole injection before preconditioning was more marked and statistically significant when the preconditioning ischemia was 2 min: the difference in %IS/AR was statistically significant between the 2′PC and DIP-2′PC groups but not between the 3′PC and DIP-3′PC groups. On the other hand, the effect of dipyridamole administration after preconditioning was modest, as shown in Fig. 5. Injection of dipyridamole after 2- and 3-min preconditioning reduced %IS/AR by only 7 and 8%, respectively. However, the importance of adenosine-receptor activation after preconditioning was indicated by the abolition of the preconditioning effect by SPT administered between 3-min preconditioning and 30-min sustained ischemia: %IS/AR in the 3′PC-SPT group was 34.5 ± 3.2% (p = NS vs. the control group).

TABLE 3
TABLE 3:
Infarct size data in experiment 2
FIG. 4
FIG. 4:
Effect of dipyridamole treatment before preconditioning ischemia. Infarct size is shown as percentage of risk zone. Abbreviations: see Fig. 1. *p < 0.05 vs. control.
FIG. 5
FIG. 5:
Effect of dipyridamole treatment after preconditioning ischemia. Infarct size is shown as percentage of risk zone. Abbreviations: see Fig. 1. *p < 0.05 vs. control.

DISCUSSION

In this study, 0.25 mg/kg of dipyridamole enhanced ischemia-induced increase of dialysate adenosine from the rabbit myocardium in situ. Pretreatment with the same dose of dipyridamole significantly potentiated the infarct size-limiting effect of preconditioning with 2-min ischemia, but its effect on preconditioning with 3-min ischemia was slight and statistically insignificant. On the other hand, dipyridamole administered between preconditioning and sustained ischemia did not alter the potency of preconditioning regardless of the duration of preconditioning ischemia. However, the importance of adenosine-receptor activation during sustained ischemia was indicated by the finding that an adenosine receptor blocker, SPT, administered after preconditioning, inhibited the protective effect of preconditioning against infarction. These results suggest that the interstitial adenosine level during preconditioning ischemia primarily determines the potency of cardioprotection by preconditioning, and that a slight increase in the adenosine level may be necessary fully to activate the adenosine-induced protective mechanism during ischemic insult.

Effect of dipyridamole on interstitial adenosine: assessment by in vivo microdialysis

Adenosine receptors in the cardiomyocytes are activated by adenosine in the cardiac interstitium. Several methods have been developed to assess the level of interstitial adenosine in the heart, including analysis of epicardial transudate (21), epicardial disk method (22), calculation from arterial and venous adenosine levels (23,24), and microdialysis (13-15,25,26). The microdialysis technique allows analysis of the temporal profile of interstitial adenosine in vivo in a more direct manner than the assay of adenosine in coronary arterial and venous blood. Furthermore, the baseline adenosine level estimated by the microdialysis technique in this and other studies is consistent with the value recently estimated by Kroll and Stepp (24) from coronary artery and venous adenosine by using a multiple-indicator dilution technique. However, a shortcoming in the in vivo microdialysis technique is a relatively large variation in the purine data on an animal-to-animal basis. There are two possible causes for this variation in the data: the location of the probe within the left ventricular wall and the extent of tissue injury caused by insertion of the probe. The interstitial adenosine level is suggested to be higher under a non-ischemic condition (27), and coronary occlusion caused more severe ischemia in the subendocardial zone than in the subepicardial zone (28,29). Thus when the microdialysis probe is placed closer to the endocardium, the adenosine level is expected to be higher.

The dialysate adenosine level is probably also modified by the tissue injury around the dialysis probe. Some mechanical tissue injury is inevitably caused by insertion of the probe into the ventricular wall, and the accumulation of blood clots and tissue edema around the dialysis probe would disturb diffusion of purines into the perfusate in the dialysis probe. Nevertheless, individual variations in the dialysate purine data could mask the effects of any drugs to modify the interstitial purine level. Accordingly, critically to assess the effect of dipyridamole on interstitial adenosine, we tried to select rabbits that showed a comparable increase of dialysate adenosine during ischemia. For this purpose, in Experiment 1, we set up a test episode of 2-min ischemia and 33-min washout period before the dipyridamole (or vehicle) challenge. A problem in this approach is that ischemia-induced increase of dialysate adenosine is attenuated after the myocardium received the first episode of ischemia. However, this alteration in interstitial adenosine would not greatly compromise the assessment of the effect of dipyridamole, because the effect of the first test episode of ischemia on tissue purines is modest compared with that after a longer episode of ischemia (13-15) and should be comparable between dipyridamole-treated and -nontreated groups. As shown in Fig. 3, the level of adenosine during ischemia after the dipyridamole treatment was 70% higher than that after vehicle treatment, indicating that dipyridamole is capable of enhancing the increase of the interstitial adenosine level during ischemia in the rabbit heart in situ.

If dipyridamole inhibits the uptake of adenosine into myocytes and endothelial cells, the interstitial adenosine level would be increased, whereas the inosine and hypoxanthine levels are expected to be reduced because these purines are catabolites of adenosine. The dialysate adenosine level during ischemia was indeed increased by dipyridamole, but the levels of inosine and hypoxanthine were not statistically different between dipyridamole-treated and -untreated rabbits (Fig. 3). In a recent study by Wang et al. (30), the dialysate inosine level was unchanged, and the hypoxanthine level was significantly reduced by dipyridamole (0.5 mg/kg, i.v.) in the canine heart, whereas the dialysate adenosine level was significantly increased. They proposed the possibility that the absence of changes in the dialysate inosine level may be caused by an alternative pathway of inosine production (i.e., dephosphorylation of inosine 5′-monophosphate) and multiple compartments with adenosine deaminase. The reason for the small reduction in the hypoxanthine level in our study is not clear. However, it is possible that the alteration of adenosine by dipyridamole was rather small in this experiment, resulting in an undetectable change in the hypoxanthine level. In addition, a possible difference in the activities of ecto-5′-nucleotidase and purine phosphorylase between the dog and rabbit hearts (23,31) might also contribute to the different results between the study by Wang et al. (30) and our study.

Preconditioning with 2-min ischemia versus preconditioning with 3-min ischemia: difference in protective effect and in enhancement by dipyridamole

In this study, preconditioning with 2-min ischemia and that with 3-min ischemia limited infarct size to 78 and 50% of the control value, respectively (Table 3). This difference in the protection of 2-min and 3-min preconditioning confirmed previous findings (6,7) that the preconditioning effect depends on the duration of preconditioning ischemia. The extent of myocardial salvage by preconditioning increased by prolonging the duration of preconditioning ischemia from 2 to 5 min (6), but no further protection was afforded by extending preconditioning ischemia to 15 min (7). It should also be noted that myocardial salvage by preconditioning results from protection of the mid-subepicardial region. A possible explanation for these findings is that the myocardial area, where the interstitial adenosine increases to a sufficient level for triggering preconditioning, spreads from the subendocardial zone to the subepicardial zone during preconditioning ischemia. If this is indeed the case, the mid-subepicardial region of the myocardium would be unprotected when preconditioning ischemia is short because of insufficient adenosine-receptor activation, resulting in less myocardial salvage. This possibility is supported by two lines of evidence. First, the adenosine level in the dialysate from the ischemic myocardium 2 min after coronary occlusion was ∼0.15 μM(Fig. 3), which is only 50% of the change observed after 5-min ischemia in the same rabbit model (15). Second, it has been shown that the interstitial adenosine level is higher in the subendocardium than in the subepicardium during ischemia (27).

We previously reported that administration of nucleoside-transport inhibitors before preconditioning with 2-min ischemia significantly potentiates the cardioprotection of preconditioning (19,20). In those studies, we could not differentiate whether the effect of the nucleoside-transport inhibitor is through its action during preconditioning ischemia or that during sustained ischemia. Our study showed that enhancement of the infarct size-limiting effect of dipyridamole was best observed when the preconditioning ischemia was 2 min and when this agent is given before preconditioning (Fig. 4). When dipyridamole was given after preconditioning or when the preconditioning ischemia was extended to 3 min, the potentiation of preconditioning was very modest and statistically insignificant (Figs. 4 and 5). These results suggest that dipyridamole reduces the temporary threshold of preconditioning ischemia, and that the potency of preconditioning protection is determined by the rate or duration of the interstitial adenosine-level increase during the preconditioning procedure and not during sustained ischemia.

However, failure of dipyridamole given after preconditioning to enhance preconditioning protection does not necessarily argue against the importance of adenosine-receptor activation during ischemic insult. Interstitial adenosine in the heart increases above the level of Kd of the adenosine receptors (32) without the nucleosidetransport inhibitor (13-15), although the rate of adenosine-level increase would be slower. Moreover, infarct-size limitation by 3-min preconditioning was completely prevented by SPT, an adenosine-receptor antagonist, administered between preconditioning and sustained ischemia (Fig. 5). These results suggest that the blockade of adenosine receptors during sustained ischemia inhibited cardioprotection of preconditioning, because the same dose of SPT does not modify infarct size in the non-preconditioned rabbit heart (18). The blockade of preconditioning by SPT in our study is consistent with a report by Thornton et al. (11) using a different nonselective adenosine-receptor blocker, PD115,199. In their study, PD115,199 blocked infarct-size limitation by preconditioning with 5-min ischemia, not only when this adenosine-receptor antagonist was given before preconditioning but also when it was administered between preconditioning and sustained ischemia. However, cardioprotection of preconditioning persisted when PD115,199 was administered after reperfusion after sustained ischemia. Taken together, these findings support that activation of the adenosine receptor during sustained ischemia is important for both 3-min and 5-min preconditioning to be cardioprotective.

In light of the protein kinase C hypothesis of preconditioning (2-5,10), these results shown in Fig. 5 are intriguing. The protein kinase C hypothesis proposes that this kinase is upregulated by activation of trigger mechanisms (including activation of adenosine and bradykinin receptors) during preconditioning, and that myocardial tolerance is afforded by more efficient phosphorylation of a protein (or proteins) by the upregulated protein kinase C at the time of ischemic insult. Unfortunately, conflicting results were reported in the literature regarding the alteration of protein kinase C activity by preconditioning (33-35), but the protein kinase C hypothesis can explain our findings shown in Figs. 4 and 5. According to this hypothesis, minimal activation of the adenosine receptor would be necessary during sustained ischemia for enhancing myocardial ischemic tolerance, because protein kinase C is already upregulated by preconditioning. This expectation is consistent with our results (Fig. 5), suggesting that cardioprotection of preconditioning is not enhanced by an augmented increase in the interstitial adenosine level during sustained ischemia but that activation of adenosine receptors is necessary for the cardioprotection.

In conclusion, administration of dipyridamole before preconditioning at a dose sufficient to augment ischemia-induced increase of interstitial adenosine reduced the threshold of preconditioning ischemia. The same dose of dipyridamole injected after preconditioning failed significantly to enhance preconditioning, although the importance of adenosine-receptor activation during sustained ischemia was indicated by the abolition of preconditioning by SPT administered after preconditioning. Thus it is likely that the level of interstitial adenosine during preconditioning is a primary determinant of cardioprotection afforded by preconditioning and that a slight increase in interstitial adenosine during sustained ischemia is sufficient for preconditioning to be cardioprotective. These findings are consistent with the protein kinase C hypothesis for preconditioning, which proposes upregulation of this kinase after preconditioning.

Acknowledgment: This study was supported in part by Grant-in-Aid for Scientific Research (04670547 and 08670812) from the Ministry of Education, Science and Culture, Japan.

REFERENCES

1. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 1986;74:1124-36.
2. Downey JM, Miura T. The role of adenosine in ischemic preconditioning. In: Hori M, Maruyama Y, Reneman RS, eds. Cardiac adaptation and failure. Tokyo: Springer-Verlag, 1994:147-66.
3. Sakamoto J, Miura T, Goto M, Iimura O. Limitation of myocardial infarct size by adenosine A1 receptor activation is abolished by protein kinase C inhibitors in the rabbit. Cardiovasc Res 1995;29:682-8.
4. Ytrehus K, Liu Y, Downey JM. Preconditioning protects ischemic rabbit heart by protein kinase C activation. Am J Physiol 1994; 266:H1145-52.
5. Speechy-Dick ME, Mocanu MM, Yellon DM. Protein kinase C: its role in ischemic preconditioning in the rat. Circ Res 1994;75:586-90.
6. Miura T, Goto M, Miki T, Sakamoto J, Shimamoto K, Iimura O. Glibenclamide, a blocker of ATP-sensitive potassium channels, abolishes infarct size limitation by preconditioning in rabbits anesthetized with xylazine/pentobarbital but not with pentobarbital alone. J Cardiovasc Pharmacol 1995;25:531-8.
7. Yamasaki K, Tanaka M, Yokota R, Miyamac M, Sasayama S. Preconditioning with 15-min ischemia extends myocardial infarct size after subsequent 30-min ischemia in rabbits [Abstract]. J Mol Cell Cardiol 1994;26:CCXIV.
8. Van Winkle DM, Thornton J, 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;2:613-9.
9. Wall TM, Sheehy R, Hartman JC. Role of bradykinin in myocardial preconditioning. J Pharmacol Exp Ther 1994;270:681-9.
10. 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;77:611-21.
11. Thornton J, Thornton CS, Downey JM. Effect of adenosine receptor blockade: preventing protective preconditioning depends on time of initiation. Am J Physiol 1993;265:H504-8.
12. 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;75:576-85.
13. Van Wylen DGL. Effect of ischemic preconditioning on interstitial purine metabolite and lactate accumulation during myocardial ischemia. Circulation 1994;89:2283-9.
14. Lasley RD, Konyn PJ, Hegge JO, Mentzer Jr RM. Effects of ischemic and adenosine preconditioning on interstitial fluid adenosine and myocardial infarct size. Am J Physiol 1995;269:HI460-6.
15. Miura T, Suzuki K, Shimamoto K, Iimura O. Suppression of the degradation of adenine nucleotides during ischemia may not be a sufficient mechanism for infarct size limitation by preconditioning. Basic Res Cardiol 1996;91:425-32.
16. Yanagi K. Distortion free digital low pass filter for chromatography. In: Doyama M, Kihara J, Tanaka M, Yamamoto R, eds. Computer aided innovation of new materials II. Amsterdam: Elsevier Science B.V., 1993:939-40.
17. Nachlas NM, Schnitka TK. Macroscopic identification of early myocardial infarction by alterations in dehydrogenase activity. Am J Pathol 1963;42:379-405.
18. 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 the rabbit heart. Circulation 1991;84:350-6.
19. Miura T, Ogawa T, Iwamoto T, Shimamoto K, Iimura O. Dipyridamole potentiates the myocardial infarct size-limiting effect of ischemic preconditioning. Circulation 1992;86:979-85.
20. Itoya M, Miura T, Sakamoto J, Urabe K, Iimura O. Nucleoside transport inhibitors enhance the infarct size-limiting effect of ischemic preconditioning. J Cardiovasc Pharmacol 1994;24:846-52.
21. Knabb RM, Gidday JM, Ely SW, Rubio R, Berne RM. Effects of dipyridamole on myocardial adenosine and active hyperemia. Am J Physiol 1984;247:H804-10.
22. Tietjan CS, Tribble CG, Gidday JM, et al. Interstitial adenosine in guinea pig hearts: an index obtained by epicardial disks. Am J Physiol 1990;259:III471-6.
23. Kitakaze M, Hori M, Takashima S, Sato H, Inoue M, Kamada T. Ischemic preconditioning increases adenosine release and 5′-nucleotidase activity during myocardial ischemia and reperfusion in dogs: implications for myocardial salvage. Circulation 1993;87:208-15.
24. Kroll K, Stepp DW. Adenosine kinetics in canine coronary circulation. Am J Physiol 1996;270:H1469-83.
25. Van Wylen DGL, Willis J, Sodhi J, Weiss RJ, Lasley RD, Mentzer RM Jr. Cardiac microdialysis to measure interstitial adenosine and coronary blood flow. Am J Physiol 1990;259:H1642-8.
26. Wikstrom G, Ronquist G, Waldenstrom A. Dynamics of myocardial metabolism in the preconditioned porcine heart studied using continuous microdialysis. Eur Heart J 1995;16:563-9.
27. Delyani JA, Van Wylen DGL. Endocardial and epicardial interstitial purines and lactate during graded ischemia. Am J Physiol 1994;266:H1019-26.
28. Reimer KA, Jennings RB. The "wavefront phenomenon" of myocardial ischemic death. II. Transmural progression of necrosis within the framework of ischemic bed size (myocardium at risk) and collateral flow. Lab Invest 1979;40:633-44.
29. Jugdutt BI, Hutchins GM, Bulkley BH, Becker LC. Myocardial infarction in the conscious dog: three-dimensional mapping of infarct, collateral flow and region at risk. Circulation 1979;60:1141-50.
30. Wang T, Mentzer RM Jr, Van Wylen DGL. Interstitial adenosine with dipyridamole: effect of adenosine receptor blockade and adenosine deaminase. Am J Physiol 1992;263:H552-8.
31. Borgers M, Thone F. Species differences in adenosine metabolic sites in the heart. Histochem J 1992;24:445-52.
32. Tucker AL, Linden J. Cloned receptors and cardiovascular responses to adenosine. Cardiovasc Res 1993;27:62-7.
33. Przyklenk K, Sussman MA, Simkhovich BZ, Kloner RA. Does ischemic preconditioning trigger translocation of protein kinase C in the canine heart? Circulation 1995;92:1546-57.
34. 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.
35. Prasad MR, Jones RM. Enhanced membrane protein kinase C activity in myocardial ischemia. Basic Res Cardiol 1992;87:19-26.
36. Vogt A, Barancik M, Weihrauch D, Arras M, Podzuweit T, Schaper W. Protein kinase C inhibitors reduce infarct size in pig hearts in vivo [Abstract]. Circulation 1994;90(suppl I):I-647.
    Keywords:

    Adenosine; Preconditioning; Infarct size; Dipyridamole

    © Lippincott-Raven Publishers