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1 Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710; 2 Division of Basic Medical Sciences, Mercer University School of Medicine, Macon, Georgia 31207; and 3 Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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We tested the hypothesis that activation of the 12-lipoxygenase (12-LO) pathway of arachidonic acid metabolism contributes to the protective effect of protein kinase C (PKC) activation and ischemic preconditioning (PC), and we report, in perfused rat heart, that both PC and the PKC activator 1,2-dioctanoyl-sn-glycerol (DOG) confer a similar protective effect and stimulate a comparable accumulation of 12-LO metabolites. The 12-LO product, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], was increased in DOG-treated (22.8 ± 4.4 ng/g wet wt) and PC hearts (26.8 ± 5.5 ng/g wet wt) compared with control (13.8 ± 2.1 ng/g wet wt, P < 0.05), and this increase was blocked by 12-LO or PKC inhibitors. Both DOG pretreatment and PC improved recovery of left ventricular developed pressure (LVDP) nearly twofold after 20 min of ischemia; this improvement was blocked by 12-LO inhibitors and was mimicked by infusion of 12-hydroperoxyeicosatetraenoic acid [12(S)-HpETE; 67 ± 6% recovery of LVDP vs. 35 ± 3% for untreated hearts]. Also, the protection afforded by 12(S)-HpETE, as well as by PC, was attenuated by the K+-channel blocker 5-hydroxydecanoate, suggesting that the downstream mechanisms of 12(S)-HpETE-mediated protection are similar to PC. Furthermore, PC stimulates 12-LO metabolism in perfused rabbit heart, and 12-LO inhibition blocks PC-induced cardioprotection. Thus the data suggest that 12-LO metabolism plays an important role in cardioprotection.
12-lipoxygenase; 12(S)-hydroperoxyeicosatetraenoic acid; eicosanoids
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ISCHEMIC PRECONDITIONING (PC) is a phenomenon by which the heart is rendered more tolerant to subsequent ischemia-reperfusion injury by one or more brief episodes of ischemia-reperfusion. Numerous protective effects, including a reduction of infarct size, stunning, and arrhythmias, have been observed in various animal species (9, 13, 14, 19, 31, 34). However, the mechanism(s) responsible for the cardioprotective effects of ischemic PC are unclear. One possible mechanism could involve production of metabolites of arachidonic acid during the brief periods of ischemia and reperfusion. Arachidonic acid, which is released during ischemia (4) or during ATP depletion induced by metabolic inhibition (5), can be metabolized via several enzyme systems (cyclooxygenase, 5-, 12-, and 15-lipoxygenase, and cytochrome P-450 epoxygenase) (20, 23, 33) to signaling molecules collectively known as eicosanoids, some of which have been detected in ischemic myocardium (10, 11, 16). Previous work has shown that the 12-lipoxygenase (12-LO) pathway of arachidonic acid metabolism is stimulated in myocardium by hypoxia or ischemia (12, 16, 18), and we have suggested recently (18) that activation of the 12-LO pathway might be involved in the protective effects of PC.
If 12-LO metabolites are generally important in cardioprotection, then one might expect that agents that mimic PC might also activate the 12-LO pathway. Furthermore, it might be possible to block the protective effects of agents that mimic PC with 12-LO inhibitors.
It has been suggested that activation of protein kinase C (PKC) is an early step in the PC pathway that confers the protective effect. Since the protective effect of PC can be mimicked by PKC activators (3, 17, 29, 35), the first aim of the present study is to establish whether PKC activation directly stimulates the 12-LO pathway, resulting in production of 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE]. If 12-LO metabolism is stimulated by PKC activation and PC, then we wanted to determine whether the beneficial effect would be eliminated by the 12-LO inhibitor baicalein at concentrations that block the production of 12(S)-HETE. We further tested whether the PC-induced stimulation of 12-LO metabolism and cardioprotection were attenuated by inhibitors of PKC. Finally, we wanted to see whether we could mimic the protective effect of PKC activation and ischemic PC by direct administration of 12-hydroperoxyeicosatetraenoic acid [12(S)-HpETE], the immediate product of the 12-LO reaction, and if so, whether the effect was due to 12(S)-HpETE itself or a downstream metabolite, by comparing the effects of 12(S)-HpETE and 12(S)-HETE. The data show that 12-LO metabolism is stimulated by PC and PKC activation protocols that protect against postischemic contractile dysfunction, that the protective effects can be eliminated by 12-LO inhibitors in parallel with elimination of the production of 12-LO metabolites, and that the protective effect can be mimicked by direct administration of 12(S)-HpETE, providing strong evidence that the 12-LO pathway of arachidonic acid metabolism is involved in the protective effect of ischemic PC and PKC activation. We further show that this pathway is important in PC in both rat and rabbit heart.
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MATERIALS AND METHODS |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Isolated Rat and Rabbit Heart Preparation
In this study, all animals received humane care in accordance with National Institutes of Health (NIH) "Guide for the Care and Use of Laboratory Animals" [DHHS Publication No. (NIH) 85-23, Revised 1985, Office of Science and Health Reports, Bethesda, MD 20892]. Male Sprague-Dawley rats (170-350 g) were anesthetized with intraperitoneal pentobarbital (~25 mg). Male New Zealand White rabbits (1-1.5 kg) were anesthetized by intravenous injection of pentobarbital (~100 mg) into a marginal ear vein. The animals were heparinized (200 U iv), the hearts were rapidly excised, and the aortas were cannulated. Retrograde perfusion was begun under constant pressure (90 cmH2O). The nonrecirculating perfusate was a Krebs-Henseleit buffer containing (in mM) 120 NaCl, 4.7 KCl, 1.2 MgSO4, 1.25 CaCl2, 25 NaHCO3, and 11 glucose. The buffer was maintained at pH 7.4 by bubbling with a mixture of 95% O2-5% CO2, at a temperature of 37°C.To monitor contractility, a latex balloon connected to a Statham pressure transducer was inserted into the left ventricle. The balloon was inflated to give an end-diastolic pressure of ~10 cmH2O. Global ischemia was created by cross-clamping the perfusate inflow line. To minimize "no-reflow" at the end of the ischemic period, the balloon was collapsed when the heart was reperfused. After a few minutes of reperfusion, the balloon was reinflated to an end-diastolic pressure of ~10 cmH2O to assess recovery of contractile function.
Biochemical Assay for 12(S)-HETE
Hearts were frozen in liquid nitrogen and stored in liquid nitrogen until extracted. 12(S)-HETE was extracted by the method described previously (30). After chloroform-methanol extraction, the chloroform phase was dried under nitrogen gas, purified with a C18 column, and stored at
70°C until assayed. The recovery was
calculated using
[14C]14,15-epoxyeicosatrienoic
([14C]14,15-EET),
which was coeluted on the column. The extracted 12(S)-HETE was quantitated using an
enzyme immunoassay kit (PerSeptive Diagnostics, catalog no. 8-6812).
The 12(S)-HETE content in the heart
is expressed as nanograms per gram of wet weight.
Experimental Protocols
Perfusion protocols for the rat heart experiments are illustrated in Fig. 1. The protocol consisted of a 20-min equilibration period, a treatment period, a 20-min period of global normothermic ischemia, and a 20-min reperfusion period. The differences in the treatment period are summarized as follows. In the control protocol, hearts were perfused with Krebs-Henseleit buffer for 25 min. In the 1,2-dioctanoyl-sn-glycerol (DOG) protocol, hearts were perfused with Krebs-Henseleit buffer for 10 min, and then 3 µM DOG was added for 10 min, followed by a 5-min washout period. In the DOG + inhibitor protocol, an inhibitor (10 µM baicalein) was perfused for 25 min beginning 10 min before the addition of DOG, throughout the 10-min period with DOG, and during the 5-min DOG-washout period; the DOG exposure was identical to the DOG protocol group. In the PC protocol, hearts were preconditioned with four cycles of 5 min of ischemia each separated by 5 min of reflow; the PC + inhibitor protocol was identical to that for the PC group except that an inhibitor [10 µM baicalein, 20 µM phenidone, 2 µM chelerythrine, or 100 µM 5-hydroxydecanoate (5-HD)] was added to the perfusate 10 min before PC and was present throughout the PC protocol. In the "inhibitor alone" protocol, an inhibitor (10 µM baicalein, 20 µM phenidone, 2 µM chelerythrine, or 100 µM 5-HD) was added to the perfusate for 25 min. In the 12(S)-HpETE group, the hearts were perfused for 5 min with Krebs-Henseleit buffer followed by perfusion with 0.4 µM 12(S)-HpETE for 10 min followed by a 5-min washout period. In the 12(S)-HpETE + 5-HD group, 100 µM 5-HD was perfused for 20 min beginning 5 min before the addition of 12(S)-HpETE, throughout the 10-min period with 0.4 µM 12(S)-HpETE, and during the 5-min 12(S)-HpETE washout period. In the 12(S)-HETE group, hearts were perfused for 5 min with Krebs-Henseleit buffer, followed by perfusion with 12(S)-HETE for 10 min, followed by a 5-min washout period.
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The protocol for rabbits was identical to that for rats, except that the ischemic period was 30 min followed by a 30-min period of reperfusion.
Left ventricular developed pressure and 31P nuclear magnetic resonance. These studies were performed in the nuclear magnetic resonance (NMR) spectrometer to monitor high-energy phosphates as described previously (3, 18, 31). Left ventricular developed pressure (LVDP) was monitored continuously. For rat hearts, we report recovery of LVDP measured at 20 min of reflow, as a percentage of the preischemic LVDP, before PC or drug administration, as the index of the protective effect. For rabbit hearts, recovery of LVDP was measured at 30 min of reflow.
LVDP with 12(S)-HpETE. Because of the lability of 12(S)-HpETE, this compound was added as a 10× stock, infused at 1/10th of the coronary flow rate, directly above the aorta via polyethylene tubing connected to a Harvard pump. We also wanted to minimize the dead space in the perfusion apparatus, and therefore these experiments were not performed in the NMR spectrometer. Several preliminary studies were performed to determine whether a concentration of 12(S)-HpETE could be found that would have an effect on recovery of function after 20 min of global ischemia. We found that 0.4 µM was an effective concentration, and this was used for subsequent studies. 12(S)-HETE content. The hearts (rabbit or rat) were freeze-clamped using tongs precooled with liquid nitrogen at the time indicated in Fig. 1. The treatment protocols were the same as described in Fig. 1, except that the protocols were terminated early. Tissue 12(S)-HETE content was measured as follows: in control hearts, after 25 min of control perfusion; in DOG-treated hearts, at the end of 10 min of DOG perfusion; in DOG + baicalein-treated hearts, at the end of 10 min of DOG perfusion (in the presence of baicalein); in PC hearts, at the end of the second 5-min period of ischemia; in PC + baicalein- or PC + chelerythrine-treated hearts, at the end of the second 5-min period of ischemia; and in baicalein- or chelerythrine-treated hearts, after 20 min of perfusion with baicalein.Materials
DOG (Sigma, St. Louis, MO) and baicalein (Calbiochem) were dissolved in DMSO and diluted to their final concentrations with perfusate immediately before use. Phenidone (Sigma) was dissolved in ethanol and diluted in perfusate immediately before use. The final concentration of ethanol or DMSO was <0.1%. Chelerythrine (Calbiochem) was dissolved in distilled water and diluted into perfusate immediately before use. 5-HD (Research Biochemicals International) was dissolved directly in the perfusate to a final concentration of 100 µM. 12(S)-HpETE and 12(S)-HETE (Cayman Chemical) were obtained in ethanol, protected from light, and diluted into the perfusate as described.Statistics
Values are expressed as means ± SE. Statistical analyses were performed by a Systat 5 program using fully factorial (M)ANOVA. When the ANOVA demonstrated that significant differences existed, a post hoc test (Fisher) was performed. The level of statistical significance was P < 0.05.| |
RESULTS |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Effects of Interventions on Hemodynamics Before Ischemia
There were no significant differences in baseline LVDP, heart rate, or coronary flow between experimental groups (see Table 1 for rat heart data and Table 2 for rabbit heart data). No significant hemodynamic effects were observed with the PKC activator DOG, the 12-LO inhibitors baicalein and phenidone, the K+-channel inhibitor 5-HD, and several 12-LO metabolites. There were no significant differences in LVDP, heart rate, or coronary flow rate among the groups at the end of the control period. None of the agents modified heart rate or coronary flow significantly. Neither DOG, baicalein, 5-HD, 12(S)-HpETE, nor 12(S)-HETE had any significant effect on LVDP. As observed previously (31), ischemic PC resulted in a decline in LVDP to 72% of the initial value at the end of the fourth reflow, before the sustained period of ischemia. This decrease in LVDP in hearts preconditioned in the presence of inhibitors was similar to the decrease in LVDP with PC alone. Pretreatment with 12(S)-HpETE, 12(S)-HETE, and 5-HD did not significantly affect contractility or coronary flow.
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Effects of 12-LO Inhibitors on Cardioprotection Induced by PC and PKC Activation
Figure 2 shows that PC improved postischemic LVDP recovery (as a percentage of initial) at 20 min of reperfusion after 20 min of ischemia (88 ± 3% vs. 47 ± 3% in control, P < 0.05), and that this protective effect was eliminated in hearts preconditioned in the presence of the 12-LO inhibitors baicalein (50 ± 6%, P < 0.05 vs. PC alone) and phenidone (42 ± 6%, P < 0.05 vs. PC alone), whereas baicalein and phenidone by themselves had no significant effect on postischemic functional recovery (37 ± 9% and 42 ± 6%, respectively, P > 0.05 vs. control).
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Since previous studies have suggested that the protective effects of PC might be linked to the activation of PKC (3, 17, 22, 29, 35), to the activation of the 12-LO pathway of arachidonic acid metabolism (18), and to a 5-HD-inhibitable pathway, presumably a KATP channel (1, 6, 15, 27, 28), we have attempted to order these events. The hypothesis that the protective effect afforded by PKC activation is related to 12-LO activation predicts that the protective effect of PKC activation would be attenuated by 12-LO inhibition. We found (Fig. 2) that PKC activation by DOG improved postischemic LVDP recovery (as % of initial) at 20 min of reperfusion after 20 min of ischemia (77 ± 7% vs. 47 ± 3% in control untreated hearts, P < 0.05), which was not significantly different from that in PC hearts (P = 0.725, PC vs. DOG). This improvement was abolished when the hearts were perfused with DOG in the presence of the 12-LO inhibitor baicalein (42 ± 9%, P < 0.05 vs. DOG alone).
To determine whether 12-LO activation is important for cardioprotection
afforded by PC in other species, such as in rabbit, we examined whether
PC improved postischemic functional recovery in rabbit hearts and
whether the protective effect could be blocked by 12-LO inhibition. As
shown in Fig. 3, PC enhanced
postischemic LVDP recovery at 30 min of reflow after 30 min of
ischemia (67 ± 3% vs. 50 ± 3% in control,
P < 0.05). This improvement was
eliminated when PC was in the presence of baicalein (46 ± 4%,
P < 0.05 vs. PC alone).
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Effects of Interventions on High-Energy Phosphates, Coronary Flow, and Heart Rate
Compared with the effects of the interventions on recovery of contractile function, the effects on other parameters of ischemic injury were modest. There were no significant differences in ATP content at the end of 20 min of ischemia among the experimental groups. Upon reperfusion, ATP recovered to 20-40% of initial in all groups. Creatine phosphate contents were restored nearly to their preischemic levels during reperfusion, indicating that tissue oxygenation was adequate and that aerobic metabolism had resumed in all experimental groups. In rat heart (Table 3), heart rate during reperfusion returned to 74-95% of the initial value, with no significant differences between groups. Recovery of coronary flow during reperfusion was also nearly complete (68-89%). Small but statistically significant differences in recovery of coronary flow were observed, with PC and DOG pretreatment resulting in a 20% improvement in recovery of coronary flow relative to control. In rabbit heart (Table 4), heart rate recovered comparably in all groups. Similar to its effect in rat heart, PC resulted in a 13% improvement in recovery of coronary flow in rabbit heart.
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Stimulation of 12-LO Metabolite Production by PC and PKC Activation
Although the inhibitor studies are consistent with our hypotheses, a more direct test of the hypotheses requires measurement of 12-LO metabolites, to show that the 12-LO pathway is stimulated by PC and by DOG and that the pathway is blocked by the inhibitor treatment protocols. We measured 12-LO activity by quantitating its stable end product, 12(S)-HETE (Table 5). We observed that rat hearts treated with DOG have an increased content of 12(S)-HETE compared with untreated control hearts (22.8 ± 4.4 vs. 13.8 ± 2.1 ng/g wet wt, P < 0.05); the increase is similar in the PC hearts (26.8 ± 5.5 ng/g wet wt, P > 0.05). Furthermore, the increases in 12(S)-HETE in DOG and PC hearts were attenuated by baicalein (9.1 ± 1.6 ng/g wet wt in DOG + baicalein-treated hearts, 10.0 ± 1.2 ng/g wet wt in PC + baicalein-treated hearts; neither value is significantly different from control). The increase in 12(S)-HETE in PC hearts was also attenuated by a selective PKC inhibitor, chelerythrine (13.1 ± 1.8 ng/g wet wt, P < 0.05 vs. PC alone), suggesting that PC activates the 12-LO pathway through activation of PKC. Baicalein (10 µM) and chelerythrine (2 µM) treatment each by themselves did not affect 12(S)-HETE content. Similar effects of PC were observed in the rabbit hearts. PC increased 12(S)-HETE (54.3 ± 8.1 vs. 24.0 ± 2.9 ng/g wet wt in control, P < 0.05), which was attenuated in the hearts preconditioned in the presence of baicalein (25.0 ± 4.3 ng/g wet wt, P < 0.05 vs. PC alone).
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Cardioprotective Effects of 12-LO Metabolites and Similarity to Ischemic PC
To test directly the hypothesis that 12-LO metabolites are cardioprotective, we perfused rat hearts with exogenous 12(S)-HETE and 12(S)-HpETE. As shown in Fig. 4, pretreatment with 12(S)-HpETE significantly improved postischemic contractile function. Hearts treated with 0.4 µM 12(S)-HpETE recovered 67 ± 6% of their preischemic function compared with 37 ± 2% for untreated hearts. Thus 12(S)-HpETE improved recovery of postischemic LVDP by 80%, comparable in magnitude to the effects of PC and DOG pretreatment. The protective effects of 12(S)-HpETE are specific for this compound, since addition of up to 3 µM 12(S)-HETE resulted in no significant improvement in postischemic recovery of LVDP. Furthermore, this finding suggests that the active metabolite is not a downstream product of the 12-LO pathway.
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If a 12-LO metabolite functions as a crucial intermediate in the signal transduction pathway of ischemic PC, then the downstream effectors of PC- and of 12(S)-HpETE-mediated protection should be the same. In this case, inhibitors of downstream effectors that block the protective effect of PC should also block the protective effect of 12(S)-HpETE. It has been reported that activation of a 5-HD-inhibitable K+ channel is important in PC (1, 6, 15, 27, 28). If a 12-LO metabolite plays a crucial role in PC, then the protective effect of both 12(S)-HpETE and PC should be affected similarly by 5-HD. We found (Fig. 4) that 5-HD attenuated the 12(S)-HpETE-mediated improvement in recovery of LVDP [44 ± 6%, P < 0.05 vs. 12(S)-HpETE alone and P > 0.05 vs. control], comparable to the effect of 5-HD on the PC-induced improvement in recovery of LVDP. 5-HD treatment by itself did not affect postischemic functional recovery in control hearts. These data demonstrate the similarity between the protective effect of PC and the protective effect of 12(S)-HpETE, as expected if a 12-LO metabolite plays a crucial role in the signal transduction pathways induced by PC.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Arachidonic acid and its metabolites have been implicated in the regulation of a variety of physiological and pathological processes. Arachidonic acid can be released from phospholipids in response to hormone or other stimulation and can be enzymatically oxidized to biologically active metabolites (eicosanoids) by several pathways. Arachidonic acid can be converted to prostaglandins and thromboxanes by the cyclooxygenase pathway, to epoxyeicosatrienoic acids by the cytochrome P-450 monooxygenase, and to 5-, 12-, and 15-hydroperoxyeicosatetraenoic acid, hydroxyeicosatetraenoic acids, leukotrienes, hepoxilins, and lipoxins by lipoxygenases. These metabolites are produced in a tissue-specific manner and have biological actions that vary by organ and species.
Using HPLC to measure lipoxygenase metabolites, we have shown previously (18) that ischemic PC resulted in a measurable increase only in 12-LO metabolites. This is consistent with studies of hypoxia and reoxygenation in isolated cardiac myocytes that demonstrated that 12(S)-HETE was the principal lipoxygenase end product that accumulated (12). In our previous study we also demonstrated that addition of inhibitors of lipoxygenase metabolism [nordihydroguariaretic acid (NDGA) and eicosatetraynoic acid (ETYA)] blocked the protective effects of PC. These previous studies (18) suggested that lipoxygenase metabolism is involved in the protection afforded by PC in rat heart. These observations have been extended in the present study by the use of the more specific 12-LO inhibitors baicalein and phenidone, which show the same ability to eliminate the protective effect of PC as NDGA and ETYA. We have now tested a total of four structurally unrelated lipoxygenase inhibitors, used at concentrations that block the 12-LO reaction, and we find that all consistently block the protective effect induced by PC. Although some aspects of the PC phenomenon appear to be different in rat myocardium than in other species, we observed that the increase in 12(S)-HETE content in rabbit myocardium during PC was similar to that we found in rat myocardium, and 12-LO inhibitors eliminate the protective effect of PC in rabbit heart the same as in rat heart. We used recovery of LVDP as our endpoint in both species, which is a global measure of ischemic injury; in the rabbit heart experiments, the duration of ischemia was 30 min, and this may have induced some lethal injury as well as stunning.
In the present study, we were also interested in testing whether
lipoxygenase metabolites, particularly 12-LO metabolites, were more
generally involved in cardioprotection. There is evidence that an
important mechanism involved in PC, at least in some species, is
activation of PKC during the brief periods of ischemia and reperfusion, and we (3) and others (17, 29) have found that a
protective effect equivalent to PC can be achieved by pretreatment with
the PKC activator DOG before a sustained period of ischemia. Furthermore, several studies (17, 29, 35) have shown that the
protective effect of PC can be eliminated with PKC inhibitors. Thus
there is strong evidence that PKC activation can play an important role
in PC and can be protective per se. To date, six PKC isoforms (
,
, 
, 
, 
, and 
) (2, 26) have been detected in heart. By
using specific antibodies to different isoforms, Mitchell et al. (17)
showed that PKC- translocates to the sarcolemma after exposure of
rat hearts to diacylglycerol or PC. Ping et. al. (22) report that
translocation of PKC- increases with the number of cycles of PC in
rabbit heart, which correlates with the protective effect. Thus,
although there is not uniform agreement on which PKC isoforms are
translocated by PC, there is general consensus that PC is associated
with translocation of only a few isoforms. Furthermore, although PKC
activators and PC have a similar protective effect, it is not clear
whether 12-LO metabolism is involved in the protection induced by PKC activators.
There are several possible ways that PC, PKC activation, and 12-LO activation could be related. It is conceivable that both PKC and 12-LO activation could occur during brief periods of ischemia and reperfusion, and both could be necessary for the protective effect of PC, but they could represent separate pathways with no direct connection. Alternatively, PKC activation could be proximal to 12-LO activation during PC, in which case DOG should increase 12-LO metabolism, and if 12-LO activation is a critical downstream effector of PKC activation, then inhibitors of lipoxygenase metabolism should block the protection afforded by DOG. Furthermore, PKC inhibition during PC should prevent the increase in 12-LO metabolites. The data in this report confirm the latter hypothesis; a significant increase in 12(S)-HETE accompanied the cardioprotection induced by DOG; 12-LO inhibition with baicalein blocked both the increase in 12(S)-HETE and the protective effect of DOG; and chelerythrine treatment during PC prevented the increase in 12(S)-HETE. Thus production of 12-LO metabolites during PC requires PKC activation, and the protective effect of PKC activation is dependent on stimulation of 12-LO. To our knowledge, this is the first report that PKC can activate 12-LO metabolism in cardiac tissue, although activators of PKC have been shown to activate phospholipase A2 in other tissues (21, 25).
In addition to showing that PKC activation can stimulate the 12-LO pathway of arachidonic acid metabolism, the present study also indicates that 12(S)-HpETE is the active metabolite responsible for the protective effect, as indicated by the following evidence. An equivalent protective effect on postischemic contractile dysfunction was seen with perfusion of 12(S)-HpETE and perfusion of DOG. Protective effects were not seen with arachidonic acid (18), suggesting that 12-LO activation is necessary for the effect, or with 12(S)-HETE, suggesting that the effect is not due to a downstream metabolite of the 12-LO pathway. We have also found protective effects of P-450 metabolites of arachidonic acid (11,12-EET), but the protective effect is not as marked as with PC or with 0.4 µM 12(S)-HpETE, even with a >10-fold greater concentration of 11,12-EET (5 µM) (32).
A final test of the hypothesis that production of 12-LO metabolites is critical for the protective effect of PC is to determine whether downstream effectors of the protective effect of PC and 12(S)-HpETE are the same. Numerous studies (1, 7, 8, 24) suggest that activation of K+ channels may function as a downstream mechanism in PC. This concept is supported by studies showing that increasing K+ channel activity can reduce myocardial ischemic injury (8) and inhibitors of K+ channels can abolish the protective effects of PC (1, 7, 24). Our data are consistent with the involvement of a K+ channel in PC, but the precise K+ channel is not defined, although recent studies suggest that the K+ channel is mitochondrial (6, 15, 27). If a 12-LO metabolite is a critical intermediate in the protective effect of PC, then a K+-channel inhibitor that can eliminate the protective effect of PC should also be able to eliminate the protective effect of 12(S)-HpETE. The data confirm this prediction; the protection afforded by 12-(S)-HpETE and PC were both blocked by the K+-channel blocker 5-HD. Thus this study allows an ordering of the mediators of PC. In rat and rabbit myocardium, PC appears to activate PKC, which leads to production of 12-LO metabolites, which directly or indirectly mediates the protective effect via a 5-HD-inhibitable K+ channel. These data are consistent with a recent report showing that PKC activators are upstream of the 5-HD-inhibitable K+ channel (27).
In summary, our data suggest that a 12-LO metabolite of arachidonic acid is involved in the signal transduction pathway responsible for cardioprotection induced by PC or PKC activation. The data satisfy Koch's postulates concerning the importance of 12(S)-HpETE in the cardioprotective effects of both PC and direct PKC activators. We have shown that the 12-LO pathway of arachidonic acid metabolism is stimulated during PC and in response to PKC activation by direct measurement of the stable metabolite of this pathway, 12(S)-HETE; we have shown that a variety of 12-LO inhibitors block the protective effect of PC or direct PKC activation on functional recovery; we have shown that a PKC inhibitor blocks the stimulation of the 12-LO pathway during PC and concomitantly eliminates the protective effect; and we have shown that we can reproduce the protective effect by direct administration of exogenous 12(S)-HpETE. The data demonstrate the central role of both PKC and 12-LO in the protective effect of PC on the improvement in postischemic functional recovery in rat heart and also indicate an important intermediary step between PKC activation and increased K+ channel activity. The data strongly suggest that 12(S)-HpETE is the active metabolite in the 12-LO pathway that is involved in the protective effect. The data also suggest that the beneficial effects of PC in animal species other than rat, such as rabbit, are associated with the activation of the 12-LO pathway.
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ACKNOWLEDGEMENTS |
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We thank Dr. Darryl Zeldin for helpful discussions and for providing [14C]14,15-EET to monitor recovery in our 12(S)-HETE assay.
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FOOTNOTES |
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Drs. W. Chen and C. Steenbergen were supported in part by National Heart, Lung, and Blood Institute Grant R01-HL-39752.
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: C. Steenbergen, Dept. of Pathology, Box 3712, Duke Univ. Medical Center, Durham, NC 27710 (E-mail: steen001{at}mc.duke.edu).
Received 16 September 1998; accepted in final form 16 February 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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P < 0.05 compared with
DOG.
