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Am J Physiol Heart Circ Physiol 276: H1229-H1235, 1999;
0363-6135/99 $5.00
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Vol. 276, Issue 4, H1229-H1235, April 1999

Importance of PKC and tyrosine kinase in single or multiple cycles of preconditioning in rat hearts

Ryan M. Fryer, Jo El J. Schultz, Anna K. Hsu, and Garrett J. Gross

Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Both tyrosine kinase (TK) and protein kinase C (PKC) inhibitors have been shown individually to completely abolish the cardioprotective effects of ischemic preconditioning (IPC) in rabbits; however, blockade of both enzymes is necessary to totally abolish IPC in pigs. Recently, we have shown that TK inhibition partially attenuates the cardioprotective effect of IPC in intact rat hearts. Therefore, the present study was designed to test the hypothesis that inhibition of both TK and PKC is necessary to completely abolish IPC in the intact rat and that this effect is dependent on the intensity of the preconditioning stimulus. All animals were subjected to 30 min of coronary artery occlusion and 2 h of reperfusion. In series 1, multiple-cycle-induced IPC was produced via three 5-min occlusions interspersed with 5 min of reperfusion (3 × 5 IPC). Genistein (5 mg/kg), a TK inhibitor infused 30 min before IPC, and chelerythrine chloride (5 mg/kg), a PKC inhibitor infused 5 min before the prolonged ischemic insult, were administered alone or in combination in the absence or presence of 3 × 5 IPC. 3 × 5 IPC produced a marked reduction in infarct size as a percentage of area at risk compared with control (8.0 ± 0.8 vs. 56.1 ± 0.8%). The effects of 3 × 5 IPC were partially blocked by pretreatment with genistein (34.0 ± 2.0%) or chelerythrine (26.4 ± 2.8%) alone; however, combined administration of genistein and chelerythrine completely abolished the effects of 3 × 5 IPC (50.7 ± 3.6%). In series 2, single-cycle IPC was elicited by one 5-min occlusion followed by 10 min of reperfusion (1 × 5 IPC). Compared with control, 1 × 5 IPC also significantly reduced infarct size (15.4 ± 3.0%). Genistein or chelerythrine administered alone completely abolished 1 × 5 IPC-induced cardioprotection. These results suggest that the efficacy of TK and PKC inhibition to block IPC depends on the intensity of the preconditioning stimulus and that these kinases may work through parallel pathways.

protein kinase C; ischemic preconditioning; genistein; chelerythrine


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

BRIEF PERIODS OF ISCHEMIA and reperfusion in the heart confer resistance to a subsequent prolonged ischemic insult (16). This phenomenon has been termed ischemic preconditioning (IPC) and has been shown to occur in dogs (16), rabbits (11), pigs (23), rats (12), and possibly humans (30, 32). Many mediators and effectors have been proposed to be essential for IPC and include the KATP channel (7, 26, 28), protein kinase C (PKC) (29, 31, 36), A1- and A3-adenosine receptors (2, 21), delta 1-opioid receptors (25), and, recently, tyrosine kinase (TK) (5, 13, 34). Although much is known about the mediators of IPC, the signaling leading to the acquisition of cardioprotection in animals has thus far remained elusive; the present study seeks to further dissect this pathway.

Inhibition of either TK (3) or PKC (35) has been shown to completely abolish IPC in the rabbit model. However, Vahlhaus et al. (33) demonstrated in the porcine model that effective attenuation of IPC required the simultaneous inhibition of both enzymes. They also suggested a complex signal cascade involving PKC and TK such that inhibition of one protein kinase may lead to a shift to an alternate, parallel pathway involving the other protein kinase, or that activation of one protein kinase may have a permissive role for the other.

A recent publication by Miura et al. (15) examined the role of PKC in the rabbit model of IPC. They found that both a single cycle [1 5-min occlusion (1 × 5)] and repetitive cycle (2 × 5) of IPC conferred equal degrees of cardioprotection and that PKC antagonists were able to attenuate IPC conferred by a single cycle of ischemia-reperfusion; however, they failed to inhibit repetitive IPC-induced cardioprotection. In addition, Sandhu et al. (22) demonstrated differences in the protection and susceptibility to PKC inhibition of single-cycle vs. multicycle transient ischemia. They demonstrated that three-cycle IPC elicited a greater protection against myocardial necrosis than one-cycle IPC and that PKC inhibition partially attenuated one-cycle IPC but did not affect IPC induced by three cycles. These data suggest that repetitive IPC may activate more pathways than a single IPC stimulus and that this difference may be attributable to the recruitment of another, PKC-independent signal transduction pathway. Recent data from our laboratory have shown that TK is involved in the initiation of IPC utilizing genistein as well as both the inactive analog of genistein, daidzein, and the structurally distinct TK inhibitor lavendustin A (5).

Therefore, on the basis of these previous results, the present study was designed to answer two major questions. 1) Do PKC or TK antagonists alone attenuate 1 × 5 and 3 × 5 IPC or is dual inhibition of PKC and TK necessary to completely abolish cardioprotection? 2) Do PKC and TK act via linear or parallel pathways and at what point in IPC is their activation important? Our data suggest that both chelerythrine and genistein completely abolish 1 × 5 IPC-induced cardioprotection, but only partially attenuate 3 × 5 IPC-induced cardioprotection when given alone. Our data also suggest that dual inhibition of both protein kinases results in complete abolishment of cardioprotection induced by 3 × 5 IPC. Last, the data suggest that TK is a proximal step in IPC in rat hearts, whereas PKC appears to be more distal and is important during the long occlusion and reperfusion period. These data further suggest that PKC and TK may act via parallel pathways to confer a potent cardioprotective effect when multiple cycles of IPC are utilized.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study was performed in accordance with the guidelines of the Animal Care Committee of the Medical College of Wisconsin, which is accredited by the American Association of Laboratory Animal Care.

General Surgical Preparation

Male Wistar rats, 350-450 g, were used for all phases of this study. The rats were anesthetized via intraperitoneal administration of Inactin (100 mg/kg), a long-acting barbiturate. A tracheotomy was performed, and the trachea was intubated with a cannula connected to a rodent ventilator (model CIV-101, Columbus Instruments, Columbus, OH, or model 683, Harvard Apparatus, South Natick, MA). The rats were ventilated with room air supplemented with O2 at 60-65 breaths/min. Atelectasis was prevented by maintaining a positive end-expiratory pressure of 5-10 mmH2O. Arterial pH, PCO2, and PO2 were monitored at control, 15 min of occlusion, and 60 and 120 min of reperfusion by a blood gas system (AVL 995 pH/blood gas analyzer) and maintained within a normal physiological range (pH 7.35-7.45, PCO2 25-40 mmHg, and PO2 80-110 mmHg) by adjusting the respiratory rate and/or the tidal volume. Body temperature was maintained at 38°C by the use of a heating pad, and bicarbonate was administered intravenously as needed to maintain arterial blood pH within normal physiological levels.

The right carotid artery was cannulated to measure blood pressure and heart rate via a Gould PE-50 or Gould PE-23 pressure transducer connected to a Grass (model 7) polygraph. The right jugular vein was cannulated for saline, bicarbonate, and drug infusion. A left thoracotomy was performed at the fifth intercostal space followed by a pericardiotomy and adjustment of the left atrial appendage to reveal the location of the left coronary artery. A ligature (6-0 prolene) was passed below the left descending vein and coronary artery from the area immediately below the left atrial appendage to the right portion of the left ventricle. The ends of the suture were threaded through a propylene tube to form a snare. The coronary artery was occluded by pulling the ends of the suture taut and clamping the snare onto the epicardial surface with a hemostat. Coronary artery occlusion was verified by epicardial cyanosis and subsequent decrease in blood pressure. Reperfusion of the heart was initiated via unclamping the hemostat and loosening the snare and was confirmed by visualizing an epicardial hyperemic response. Heart rate and blood pressure were allowed to stabilize before the following protocols were initiated.

Drugs

Inactin (thiobutabarbital sodium), genistein, and chelerythrine chloride were purchased from Research Biochemicals International, Natick, MA. 2,3,5-Triphenyltetrazolium chloride (TTC) was purchased from Sigma Chemical (St. Louis, MO). Inactin was dissolved in distilled water. Genistein was dissolved in Alkamuls EL-620 (Rhone-Poulenc), 95% EtOH, and saline. Chelerythrine was dissolved in filtered dH2O and subsequently sonicated to bring it into solution. All drugs were dissolved in ~0.9 ml vehicle for administration at all drug concentrations. Genistein vehicle did not significantly affect infarct size as a percentage of area at risk (IS/AAR) compared with control (n = 3, data not shown).

Study Groups and Experimental Protocols

The experiments were divided into two separate series. Series 1 (13 groups) examined the effect of TK and/or PKC inhibition on 3 × 5 IPC. Series 2 (9 groups, utilizing 4 of the control groups from series 1) examined the effect of either TK or PKC inhibition on 1 × 5 IPC.

Series 1. Rats were randomly assigned to 1 of 13 groups (Fig. 1). All groups underwent a 30-min coronary artery occlusion and 2-h reperfusion period after administration of genistein and/or chelerythrine in the presence or absence of IPC. The control group (Con) underwent a 30-min coronary artery occlusion and subsequent 2 h of reperfusion. The repetitive preconditioning group was subjected to a 5-min coronary artery occlusion period and 5-min reperfusion period repeated three times (3 × 5 IPC). To determine the effect of chelerythrine treatment on infarct size in control groups, three groups were administered increasing doses of chelerythrine (0.1, 1.0, and 5.0 mg/kg, respectively) 5 min before 30 min of coronary artery occlusion and 2 h of reperfusion (Che 0.1 Con, Che 1.0 Con, Che 5.0 Con). The effect of chelerythrine treatment (0.1, 1.0, and 5.0 mg/kg, respectively) on 3 × 5 IPC was assessed by administering the drug during the last 5-min reperfusion period before the prolonged ischemic insult (3 × 5 + Che 0.1, 3 × 5 + Che 1.0, 3 × 5 + Che 5.0). Similarly, the effect of pretreatment with chelerythrine 5 min before 3 × 5 IPC was also examined (Che 5.0 + 3 × 5). The TK inhibitor genistein was administered 30 min before the long occlusion and reperfusion period (Gen Con) to determine the effects of genistein on infarct size in control groups. The effect of genistein (5.0 mg/kg) on IPC was assessed by administering genistein 30 min before 3 × 5 IPC (Gen + 3 × 5). Finally, two groups were treated with both genistein and chelerythrine either in the absence or presence of 3 × 5 IPC to determine the effect of dual inhibition of both PKC and TK on infarct size. The control group was given genistein (5.0 mg/kg) 1 h before, and chelerythrine (5.0 mg/kg) 5 min before, the long occlusion and reperfusion period (Gen + Che Con). The preconditioning groups were given genistein 30 min before 3 × 5 IPC and chelerythrine (5.0 mg/kg) 5 min before the long occlusion and reperfusion period (Gen + IPC + Che).


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  		Fig. 1.   Protocol bar depicting experiments used to study effects of genistein and chelerythrine chloride on 1 5-min occlusion followed by 10-min reperfusion (1 × 5) and 3 5-min occlusions interspersed with 5 min of reperfusion (3 × 5) ischemic preconditioning (IPC) in intact rat heart. All groups underwent 30-min coronary artery occlusion (Occ) and 2-h reperfusion period. Series 1 protocols: control (Con), 30-min occlusion and 2-h reperfusion; 3 × 5 IPC, 5-min occlusion period followed by 5-min reperfusion period repeated 3 times; chelerythrine (CH) control, chelerythrine (0.1, 1.0, and 5 mg/kg) given 5 min before 30-min occlusion and 2-h reperfusion period; 3 × 5 IPC + chelerythrine, chelerythrine (0.1, 1.0, and 5.0 mg/kg) administered during last reperfusion cycle of 3 × 5 IPC; chelerythrine + 3 × 5 IPC, chelerythrine (5.0 mg/kg) administered 5 min before 3 × 5 IPC; genistein (Gen) control, genistein (5.0 mg/kg) given 30 min before 30-min occlusion and 2-h reperfusion period; genistein + 3 × 5 IPC, genistein (5.0 mg/kg) administered 30 min before 3 × 5 IPC; genistein + chelerythrine control, genistein (5.0 mg/kg) administered 1 h and chelerythrine (5.0 mg/kg) administered 5 min before control protocol; genistein + 3 × 5 IPC + chelerythrine: genistein (5.0 mg/kg) administered 30 min before 3 × 5 IPC and chelerythrine (5.0 mg/kg) administered during last reperfusion period of IPC. Series 2 protocols: 1 × 5 IPC, 5-min occlusion period followed by 10-min reperfusion period; 1 × 5 IPC + chelerythrine, chelerythrine (1.0 and 5.0 mg/kg) administered during last reperfusion cycle of 1 × 5 IPC; genistein + 1 × 5 IPC, genistein (5.0 mg/kg) administered 30 min before 1 × 5 IPC.

Series 2. Rats were randomly assigned to one of four additional protocols (Fig. 1), and these results were compared with four of the control groups utilized in series 1 (Con, Che 1.0 Con, Che 5.0 Con, Gen Con). To assess the effect of one cycle of IPC, one group was subjected to IPC established via a 5-min coronary artery occlusion and a 10-min reperfusion period (1 × 5 IPC). Chelerythrine, either 1.0 or 5.0 mg/kg, was administered during the last 5 min of reperfusion after 1 × 5 IPC before the prolonged occlusion and reperfusion period (1 × 5 + Che 1.0 and 1 × 5 + Che 5.0). Gen (5 mg/kg) was infused 30 min before 1 × 5 IPC (Gen + 1 × 5).

Determination of IS

On completion of the above protocols, the coronary artery was reoccluded and the AAR was determined by negative staining. Patent blue dye was administered via the jugular vein to effectively stain the nonoccluded area of the left ventricle. The rat was euthanized with a 15% KCl solution. The heart was excised, and the left ventricle was removed from the remaining tissue and subsequently cut into six thin cross-sectional pieces. This allowed for the delineation of the normal area, stained blue, versus the AAR, which subsequently remained pink. The AAR was excised from the nonischemic area, and the tissues were placed in separate vials and incubated for 15 min with a 1% TTC stain in 100 mM phosphate buffer (pH 7.4) at 37°C. TTC is an indicator of viable and whereas tissue. TTC is reduced by dehydrogenase enzymes present in viable myocardium resulting in a formazan precipitate and inducing a deep red color in the viable tissue whereas the infarcted area stains gray (9). Tissues were stored in vials of 10% formaldehyde overnight and the infarcted myocardium was dissected from the AAR under the illumination of a dissecting microscope (Cambridge Instruments). IS and AAR were determined by gravimetric analysis. IS was expressed as a percentage of the AAR.

Exclusion Criteria

A total of 113 rats successfully completed the above protocols. Rats were excluded from data analysis if they exhibited severe hypotension (<30 mmHg systolic blood pressure) or if we were unable to maintain adequate blood gas values within a normal physiological range due to metabolic acidosis. Exclusion of animals from the present study was evenly distributed among the protocol groups.

Statistical Analysis of Data

All values are expressed as means ± SE. Analysis of variance with Bonferroni's test was used to determine whether any significant differences existed among groups for hemodynamics, IS, and AAR. Significant differences were determined at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
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RESULTS
DISCUSSION
REFERENCES

Hemodynamics

Table 1 summarizes heart rate, mean arterial blood pressure, and rate-pressure product (RPP) in all groups (series 1 and 2) determined at baseline, at 15 min of coronary artery occlusion, and at 120 min of reperfusion. Blood pressures in the drug protocols were maintained at baseline values after genistein and chelerythrine treatment. No significant differences in baseline, 15 min of postcoronary artery occlusion, or 2 h of reperfusion in hemodynamic parameters existed between groups; however, RPP in control vs. chelerythrine (0.1 mg/kg) control at 15 min of coronary artery occlusion was significantly increased (P < 0.05).

                              
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  		Table 1.   Hemodynamic values

IS and AAR

Left ventricular weight (g), IS (g), AAR (g), IS/AAR (%), and AAR/left ventricle weight (%) for all groups, series 1 and 2, are shown in Table 2. Left ventricular weight was significantly different (P < 0.05) in 3 × 5 IPC vs. Gen Con and 1 × 5 IPC vs. Gen Con. Similarly, AAR was significantly different in 1 × 5 IPC vs. Gen + Che Con; however, there were no significant differences in AAR/left ventricle weight in the above groups.

                              
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  		Table 2.   Infarct size

Series 1. Figures 2 and 3 show IS/AAR for each group. The average IS/AAR in control rats was 56.1 ± 0.8%. 3 × 5 IPC produced a marked reduction in IS/AAR (8.0 ± 0.8%) compared with control. Genistein (5 mg/kg) and chelerythrine (0.1, 1.0, and 5.0 mg/kg) administered to nonpreconditioned rats had no significant effect on IS/AAR (52.3 ± 1.2, 52.5 ± 6.2, 48.8 ± 5.1, and 58.7 ± 6.8%, respectively). Pretreatment with genistein (5.0 mg/kg) only partially attenuated 3 × 5 IPC-induced cardioprotection (34.0 ± 2.0%). Treatment with chelerythrine (0.1, 1.0, and 5.0 mg/kg) during the final reperfusion period of 3 × 5 IPC attenuated the cardioprotection afforded via 3 × 5 IPC in a dose-dependent manner (9.5 ± 2.4, 18.8 ± 6.0, and 26.4 ± 2.8%), and this effect was significant at 5.0 mg/kg. However, treatment with chelerythrine (5.0 mg/kg) 5 min before 3 × 5 IPC did not significantly attenuate the effects of IPC (20 ± 4.8). Dual treatment with genistein and chelerythrine in control protocols did not affect IS/AAR (55.9 ± 2.0%). However, dual treatment with both genistein before IPC and chelerythrine in the last IPC cycle completely abolished the cardioprotective effects of IPC (50.7 ± 3.6%).


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  		Fig. 2.   Infarct size expressed as percentage of area at risk in rat hearts subjected to drug control, 1 × 5 IPC, 3 × 5 IPC, and either protein kinase C (PKC) or tyrosine kinase (TK) inhibitor control protocols. Chelerythrine was administered at 0.1, 1.0, and 5.0 mg/kg and genistein at 5.0 mg/kg. Drug treatment in control hearts did not affect infarct size in absence of preconditioning stimulus.


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  		Fig. 3.   Infarct size expressed as percentage of area at risk in series 1 rat hearts subjected to control, 3 × 5 IPC, and 3 × 5 IPC in presence of PKC and/or TK inhibitors. Chelerythrine exhibited a dose-dependent partial attenuation of cardioprotection when administered after 3 × 5 IPC (0.1, 1.0, and 5.0 mg/kg) but did not attenuate cardioprotection if administered before 3 × 5 IPC (5.0 mg/kg). Genistein administration before IPC partially attenuated IPC, and dual treatment with both inhibitors (genistein: 5.0 mg/kg, chelerythrine: 5.0 mg/kg) completely abolished IPC.

Series 2. Figures 2 and 4 show IS/AAR for each of the five additional groups assigned to this series as well as controls from series 1. The results of the four control groups, identical to series 1, were explained above. 1 × 5 IPC resulted in a marked reduction in IS compared with control (15.4 ± 3.0%). Pretreatment with genistein (5.0 mg/kg) or administration of chelerythrine (1.0 and 5.0 mg/kg) 5 min before the 30-min coronary artery occlusion completely abolished the cardioprotective effect of 1 × 5 IPC (41.9 ± 6.7, 45.4 ± 10.4, and 54.4 ± 8.9%, respectively).


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  		Fig. 4.   Infarct size expressed as percentage of area at risk in series 2 rat hearts subjected to control, 1 × 5 IPC, and 1 × 5 IPC in presence of either PKC or TK inhibitors. Chelerythrine (1.0 and 5.0 mg/kg) completely attenuated cardioprotection when administered after 1 × 5 IPC. Similarly, genistein (5.0 mg/kg) administered 30 min before IPC completely abolished cardioprotection.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the first series of the present study we demonstrated that inhibition of both PKC and TK was necessary to completely abolish 3 × 5 IPC-induced cardioprotection. We also demonstrated that inhibition of each protein kinase alone could only partially attenuate multiple cycles of IPC. In the second series, we demonstrated that one cycle of IPC was also effective in reducing IS; however, the protective effects of 1 × 5 IPC could be completely abolished by treatment with either PKC or TK inhibitors alone, at doses that only partially attenuated 3 × 5 IPC. We also demonstrated that treatment with the PKC antagonist chelerythrine 5 min before 3 × 5 IPC did not significantly attenuate IPC-induced cardioprotection and only antagonized IPC when given after the IPC stimulus.

Results from series 1 experiments suggest that PKC inhibition is important during the prolonged occlusion period and that one or more PKC-independent pathways may exist to confer cardioprotection induced by 3 × 5 IPC. These results are in agreement with the results of Downey's group obtained in the rabbit heart, which demonstrated that PKC activity is necessary to mediate, rather than trigger, IPC (35). Previous data from our laboratory demonstrated that genistein administered during the first or third cycle of IPC did not attenuate IPC, suggesting that TK is important in IPC during the IPC phase (5). Once again, partial attenuation of IPC with genistein pretreatment suggests that an alternate TK-independent pathway may be important to confer cardioprotection produced by 3 × 5 IPC. Because we demonstrate complete abolishment of IPC-induced cardioprotection when genistein and chelerythrine are administered during the same protocol, we suggests that cardioprotection in the in vivo rat heart subjected to multiple-cycle IPC is conferred by parallel pathways involving PKC and TK, respectively.

In the series 2 experiments, the present data suggest that 1 × 5 IPC is not as efficacious in inducing cardioprotection before a prolonged ischemic insult as 3 × 5 IPC and demonstrate that 1 × 5 IPC is easier to inhibit compared with 3 × 5 IPC. In the case of PKC-epsilon , this may be the result of less protein translocation from the cytosolic to particulate fraction, because the particulate fraction of PKC-epsilon increases in a dose-dependent fashion based on the number of occlusion-reperfusion cycles performed. These data also agree with the data previously published by Miura et al. (15) with respect to PKC inhibition. They showed that PKC inhibitors can attenuate IPC induced by one cycle, but not a repetitive cycle. Similarly, compared with 3 × 5 IPC, 1 × 5 IPC may activate less or fewer isoforms of PKC.

Classical IPC in the rat myocardium is readily accepted, and work in our laboratory suggests that the end effector of this pathway is the KATP channel (7, 26, 28). In the intact myocardium, classical IPC may be attributed to the interstitial accumulation of mediators such as adenosine (14) and opioids (27), which may activate PKC by stimulating their respective sarcolemmal receptors. Activation of PKC then may directly or indirectly affect proteins downstream. Although the mechanism of IPC remains elusive, the KATP channel may be the end effector (7), activated by Gi/o proteins (24, 25), PKC (10), and/or possibly TK.

Utilizing genistein, Maulik et al. (13) first demonstrated the importance of a TK-phospholipase D signaling pathway in classical IPC of the isolated rat heart. Recently, we demonstrated a role for TK in the initiation of IPC in the in vivo rat heart (5). However, the point at which TK is activated in IPC remains controversial. Weinbrenner et al. (34) have shown the involvement of TK later in IPC, and Baines et al. (4) suggested that TK is downstream of PKC and suggest a linear signal transduction pathway leading to IPC in rabbits. These differences may be attributed to species variation (rat vs. rabbit). In addition, genistein has also been shown to exhibit nonselective effects (1, 17, 18). Our laboratory recently suggested that the anticardioprotective effects of genistein in the in vivo rat model of IPC were due to the inhibition of TK rather than inhibition of voltage-gated Na+ channels, adenosine A1 receptors, or PKC (5). These same data demonstrated that 5 mg/kg of genistein elicited a maximal response in attenuation of IPC, because 10 mg/kg did not further attenuate IPC.

There is also discrepancy as to the effect of PKC inhibitors on IPC in the current literature, possibly due to species differences or to the dosage or to the timing of PKC inhibitor or activator administration. Indeed, Yang et al. (35) demonstrated that the protection of IPC is dependent on a critical timing of PKC activation. Our data demonstrate that chelerythrine, when administered before the prolonged ischemic event, but not when administered before IPC, could significantly attenuate 3 × 5 IPC-induced cardioprotection. These results may be due to partial metabolism of chelerythrine before the prolonged ischemic event in the latter study, thus not significantly contributing to attenuation of IPC-induced protection; however, the t1/2 of chelerythrine in the in vivo rat is unknown. Many isoforms of PKC are known to exist. Gray et al. (6) found that the activation of PKC-epsilon is critical for cardiac myocyte protection in cell culture; furthermore, Ping et al. (19) demonstrated that PKC-epsilon increased in a dose-dependent fashion as the number of occlusion-reperfusion cycles increased. This finding may explain increased protection after 3 × 5 IPC vs. 1 × 5 IPC.

Recent studies have examined the involvement of dual inhibition of PKC and TK in ischemia-reperfusion injury. Joyeux et al. (8) found, in the isolated rat heart, that heat stress-induced delayed cardioprotection required activation of PKC but not TK, and Kukreja's group (20) suggest that this delayed protection may also be attributed to an increased synthesis of both heat shock protein-27 and -72. Although IPC induced cardioprotection could be completely abolished via administration of either a PKC or TK antagonist in the rabbit model (3, 35), Vahlhaus et al. (33) recently demonstrated, in the porcine model, that dual inhibition of both PKC and TK, with staurosporine and genistein, respectively, abolished the cardioprotective effect of preconditioned animals. They suggested that blockade of either pathway results in a shift to the alternate parallel pathway, thus not attenuating IPC-induced cardioprotection. In opposition to these data, Baines et al. (4) suggested that TK is downstream from PKC in IPC-induced protection in the rabbit, suggesting a linear pathway. Our data, in agreement with Vahlhaus et al. (33), suggests a parallel pathway; however, if a linear pathway did exist, we would expect PKC to be downstream from TK because treatment with the TK inhibitor genistein only abolished cardioprotection when administered before IPC and, similarly, treatment with chelerythrine, a PKC inhibitor, only abolished cardioprotection when given during the last 5 min of IPC.

In conclusion, these data suggest that dual inhibition of PKC and TK is necessary to completely abolish 3 × 5 IPC-induced cardioprotection, but not 1 × 5-induced protection. Complete inhibition of 1 × 5 IPC by either inhibitor may result from a decreased amount of activated protein kinase, whereas only partial inhibition of 3 × 5 IPC by either inhibitor may result from a switch to an alternate, independent pathway to activate the end effector of IPC. Complete inhibition of 3 × 5 IPC via blockade of both PKC and TK suggests that these two protein kinases may exist in parallel pathways to confer cardioprotection, such that inhibition of one signal transduction pathway may cause increased transduction through the other, independent pathway. These data also suggest that PKC is important after IPC is conferred, whereas TK is important early on in the IPC cycle. Finally, the present results may also help explain the discrepancies that exist in the literature concerning the role of PKC and TK in IPC in different models, which may have resulted from using different inhibitor doses and dosing regimens.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-08311 and an advanced predoctoral fellowship from the Pharmaceutical Research and Manufacturers of America Foundation (to J. E. J. Schultz).


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: G. J. Gross, Dept. of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (E-mail: ggross{at}post.its.mcw.edu).

Received 28 September 1998; accepted in final form 15 December 1998.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 276(4):H1229-H1235
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