INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

OPIOID RECEPTOR STIMULATION is thought to be an important component of cardioprotection induced by ischemic preconditioning (IPC) (35). Interestingly, it has been demonstrated (27) that both preproenkephalin and enkephalin peptides are increased after myocardial infarction in the left ventricles (LV) of rats. In addition, opioid receptor agonists have been shown to induce cardioprotection (7, 34) and reduce the incidence of cardiac arrhythmias (12).

We (13) have previously demonstrated that cardioprotection against sustained ischemia in the rat heart following IPC or an opioid agonist is dependent on the activation of a mitogen-activated protein kinase (MAPK) cascade. Specifically, we demonstrated that cytosolic, but not nuclear, activation of extracellular signal-regulated kinase (ERK) is essential to cardioprotection. This activation is important immediately on myocardial reperfusion and is mediated by the isoforms p44 and p42 during IPC; however, cardioprotection from the -opioid agonist was dependent only on activation of p44 MAPK.

In a similar vein, both of these cardioprotective interventions may also utilize a signal transduction cascade mediated by activation of the stress-activated protein kinases p38 and c-jun NH₂-terminal kinase (JNK). However, whether activation of these enzymes is protective or detrimental to the cell is controversial (28). It has been demonstrated (3, 24, 26) that activation of either kinase may be cardioprotective and that inhibition of p38 may abolish cardioprotection in vivo. In contrast, there is an equal amount of literature suggesting that inhibition of p38 is cardioprotective in the absence of IPC. Schneider and colleagues (33) have demonstrated that inhibition of p38 with SB-202190 improved the functional recovery of isolated rat hearts following ischemia and did not abolish the recovery of preconditioned hearts. In addition, Mackay and Mochly-Rosen (21) have suggested that inhibition of this kinase may be cardioprotective via a reduction in caspase-3 activation, a key event in apoptosis. Sato and colleagues (31) have suggested that these enzymes may have different functions in animals subjected only to ischemia-reperfusion versus animals also subjected to IPC, whereby the activation of p38 and JNK may be detrimental during ischemia-reperfusion but important to trigger IPC.

Other G protein-coupled agonists have been shown to induce the activation of the MAPK signaling cascade. Haq and colleagues (16) have demonstrated in the perfused rat heart that adenosine potently induced the activation of p38 and both isoforms of JNK. In addition, they demonstrated that the p38 substrate MAPK-activated protein (MAPKAP) kinase 2 was strongly activated by adenosine. Therefore, it is a distinct possibility that opiates also induce the activation of these pathways. We hypothesize that p38 and JNK may play an important role in cardioprotection induced by IPC or ₁-opioid receptor activation in the intact rat heart.

	METHODS

TOP 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 weighing 350-450 g were used for all phases of this study. The rats were anesthetized via intraperitoneal administration of thiobutabarbital sodium (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; 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 analyzer 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 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.

Drugs. Inactin was purchased from Research Biochemical International (Natick, MA) and 2,3,5-triphenyltetrazolium chloride (TTC) was purchased from Sigma (St. Louis, MO). 2-Methyl-4a-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12a-octahydroquinolino [2,3,3-g]isoquinoline (TAN-67) was synthesized and kindly provided by Dr. Hiroshi Nagase of Toray Industries (Kanagawa, Japan) and was dissolved in saline. Inactin was dissolved in distilled water (dH₂O). SB-203580 hydrochloride was purchased from Calbiochem and dissolved in dH₂O. All drugs were dissolved in ~0.9 ml of vehicle for administration at all concentrations.

Study groups and experimental protocols. The protocols used to determine a role for p38 in cardioprotection are shown in Fig. 1. All animals were subjected to 30 min of ischemia and 2 h of reperfusion (control). The ₁-opioid agonist TAN-67 was infused 15 min before ischemia-reperfusion. IPC was induced via one cycle of a 5-min coronary artery occlusion and 5 min of reperfusion. The effect of the p38 inhibitor SB-203580 in the absence of opioid receptor stimulation or IPC was investigated by the administration of either of the two doses of this compound 25 min before the control protocol. The effect of p38 inhibition during IPC or opioid treatment was investigated via administration of a bolus of SB-203580 15 min before IPC or 10 min before TAN-67 administration. In addition, the effect of enzyme inhibition at myocardial reperfusion was investigated by administering SB-203580 (0.2 mg/kg) 5 min before reperfusion during prolonged ischemia.

View larger version (35K): [in this window] [in a new window]   Fig. 1.   Protocol bar for the determination of the importance of p38 mitogen-activated protein kinase (MAPK) in cardioprotection following ischemic preconditioning (IPC) or stimulation with the 1-opioid receptor agonist TAN-67. All animals were subjected to 30 min of regional ischemia and 2 h of reperfusion. SB-203580 (0.2 or 1.0 mg/kg) was administered 25 min before sustained ischemia in animals subjected only to ischemia-reperfusion or previously administered TAN-67 or subjected to IPC. In addition, SB-203580 (0.2 mg/kg) was administered 5 min before 2 h of reperfusion during the prolonged ischemic episode in preconditioned animals. TTC, 2,3,5-triphenyltetrazolium chloride.

Determination of infarct size. On completion of the above protocols, the coronary artery was reoccluded, and the area at risk (AAR) was determined by negative staining. Patent blue dye was administered via the jugular vein to effectively stain the nonoccluded area of the LV. The rat was euthanized with a 15% KCl solution. The heart was excised, and the LV 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 in a 1% TTC stain in 100 mM phosphate buffer (pH 7.4) at 37°C. TTC is an indicator of viable and nonviable tissue. TTC is reduced by dehydrogenase enzymes present in the myocardium, resulting in a formazan precipitate and inducing a deep red color in the viable tissue, whereas the infarcted area stains gray (18). 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). Infarct size (IS) and AAR were determined by gravimetric analysis. IS was expressed as a percentage of the AAR (IS/AAR).

Tissue sample preparation. Tissue samples were processed from the entire AAR of the LV (~350 mg) of control animals, animals treated with TAN-67, and animals subjected to IPC at 0, 5, 15, or 30 min of ischemia or 5, 30, or 60 min of reperfusion for the determination of protein expression and activity of either p38 or JNK, as previously described (13). Myocardial tissue samples, frozen at 80°C until use, were powdered with a prechilled mortar and pestle. Total cellular proteins were isolated via glass-glass homogenization of the powdered tissue in lysis buffer A containing 0.3% -mercaptoethanol, 50 mM Tris · HCl, 5 mM EDTA, 10 mM EGTA, 50 µg/ml phenylmethylsulfonyl fluoride, 200 µM sodium orthovanadate, and 1 ml/20 g tissue Sigma Protease Inhibitor Cocktail P-8340 [containing 4-(2-aminoethyl)benzenesulfonyl fluoride, pepstatin A, trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64), bestatin, leupeptin, and aprotonin in dimethyl sulfoxide]. The homogenate was separated into nuclear and cytosolic fractions by differential centrifugation as previously described (13). Briefly, the homogenate was loaded onto a sucrose cushion, containing 2 ml of 1 M sucrose in lysis buffer A, and was centrifuged at 1,600 g for 10 min to allow for pelleting of the nuclear fraction. The pellet was washed with dH₂O and resuspended in lysis buffer B (lysis buffer B contained 0.5% Igepal, 0.1% deoxycholate, and 0.1% Brij-35) for 60 min on ice and subsequently recentrifuged at 7,850 g for 5 min. The supernatant became the nuclear fraction. The supernatant from the initial centrifugation of 1,600 g was loaded onto a second 1-M sucrose cushion and was centrifuged at 150,000 g for 60 min. The supernatant became the cytosolic fraction. Total protein concentrations in the respective fractions were determined via the Bradford (Bio-Rad) protein assay. Preliminary experiments were carried out to ensure that storing the tissues at 80°C until use and powdering of the frozen tissue did not fractionate the myocardial nuclei. We (13) have previously shown that this technique specifically isolates the cytosolic from the nuclear fraction by assessing the purity of the fractions with specific antibody markers.

Western blot analysis of subcellular p38 and JNK distribution. Total (30 µg) protein from the nuclear fraction or cytosolic fraction of tissue homogenate was electrophoresed with a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. Molecular weight markers and positive controls purchased from Cell Signaling Technology (Beverly, MA) were also electrophoresed to confirm that the molecular weight of the bands were 38 or 46 and 54 kDa and for comparison between samples during densitometric analysis, respectively. The positive controls used for densitometric comparison between groups were ultraviolet-stimulated extracts from 293 cells. Electrophoresis and Western blot analysis were performed as previously described (13). Briefly, nonspecific background staining was blocked in nonfat dry milk, and the membrane was incubated with the appropriate primary antibody at a 1:1,000 dilution. The membrane was washed and incubated with the appropriate horseradish peroxidase-linked secondary antibody in blocking buffer (Bio-Rad). The membrane was washed again and stained with a chemiluminescent system (ECL kit, Amersham), and densitometry was performed on each sample and analyzed via National Institutes of Health Image Software. Phosphospecific polyclonal antibodies against p38 and JNK were purchased from New England Biolabs (Beverly, MA). The results of the Western blot analysis for JNK and p38 are expressed in arbitrary units of relative density, whereby the signal intensity of each lane is compared back to the signal intensity of the respective positive controls and expressed as a percentage of the positive control.

Exclusion criteria. A total of 55 rats successfully completed the above protocols for IS analysis. An additional 74 rats completed the above protocols for Western blot analysis. 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. One-way analysis of variance with Newman-Keuls post hoc test was used to determine whether any significant differences existed among groups for hemodynamics, IS, and AAR. Significant differences for IS and hemodynamic analysis were determined at P < 0.05. Significant differences in the relative density of the nuclear and cytosolic fractions were also determined at P < 0.05 by an unpaired Student's t-test.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Hemodynamic data and IS analysis. The hemodynamic data are summarized in Table 1. There were no consistent differences in any group versus control for HR, mean blood pressure, or rate-pressure product. However, SB-203580 significantly reduced the HR throughout the protocols in all animals except those also subjected to IPC at baseline and 15 min of ischemia. In addition, although the rate-pressure product was not altered in any group throughout the protocol, mean blood pressure was increased in IPC animals at 2 h of reperfusion and in animals administered the high dose of SB-203580 in the presence of TAN-67 at 0 min of ischemia.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Infarct size expressed as a percentage of the area at risk in each of the 8 protocols. *P < 0.05 vs. control. P < 0.05 vs. IPC.

Relative density of p38 MAPK. The relative density in the cytosolic and nuclear fractions of p38 and p46/p54 JNK in control, TAN-67-, and IPC-treated animals at baseline, 5, 15, and 30 min of ischemia and 5, 30, and 60 min of reperfusion are represented in Figs. 3-7 and were determined by the phosphorylation state of the enzyme as detected by the phospho-specific primary antibody against JNK or p38. All values are representative of 3-6 animals per treatment group per time point.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Relative density of p38 MAPK in the nuclear fraction of control (), TAN-67-treated (), or IPC () animals at baseline, 5, 15, and 30 min of ischemia or 5, 30, and 60 min of reperfusion. *P < 0.05 vs. control.

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Relative density of p38 MAPK in the cytosolic fraction of control (), TAN-67-treated (), or IPC () animals at baseline, 5, 15, and 30 min of ischemia or 5, 30, and 60 min of reperfusion. *P < 0.05 vs. control.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Relative density of p54 c-jun NH2-terminal kinase (JNK) kinase (A) and p46 JNK (B) in the nuclear fraction of control (), TAN-67-treated (), or IPC () animals at baseline, 5, 15, and 30 min of ischemia or 5, 30, and 60 min of reperfusion.

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Relative density of p54 JNK (A) or p46 JNK (B) in the cytosolic fraction of control (), TAN-67-treated (), or IPC () animals at baseline, 5, 15, and 30 min of ischemia or 5, 30, and 60 min of reperfusion. *P < 0.05 vs. control. P < 0.05 vs. TAN-67.

View larger version (58K): [in this window] [in a new window]   Fig. 7.   A: Western blot of p54 and p46 JNK in the cytosolic fraction of control (Con), TAN-67, and IPC samples at 5, 30, and 60 min of reperfusion. Ultraviolet (UV)-treated cells (n = 293 cells) served as a positive control. B: relative density of p54 JNK at 5 min of reperfusion in the cytosolic fraction shown as a bar graph in control, TAN-67, and IPC samples. C: relative density of p46 JNK at 5 min of reperfusion in the cytosolic fraction shown as a bar graph in control, TAN-67, and IPC samples.

Relative density of p54 and p46 JNK. We also examined the phosphorylation state of both isoforms, p46 and p54, of JNK throughout ischemia-reperfusion in the nuclear (Fig. 5) and cytosolic (Fig. 6 and Fig. 7) fractions. There was a slight increase in the relative density of both isoforms immediately on myocardial reperfusion in the nuclear fraction. Listed are the relative densities of p46 and p54 phosphorylation, respectively, at baseline, 5 min of reperfusion, and 60 min of reperfusion for control (0.36 ± 0.07, 1.38 ± 0.18, 0.71 ± 0.15 and 0.07 ± 0.01, 0.46 ± 0.02, 0.29 ± 0.04), TAN-67-treated animals (0.44 ± 0.14, 1.05 ± 0.02, 0.61 ± 0.04 and 0.08 ± 0.01, 0.39 ± 0.04, 0.29 ± 0.04), and animals subjected to IPC (0.53 ± 0.05, 0.94 ± 0.08, 0.60 ± 0.04 and 0.10 ± 0.02, 0.29 ± 0.02, 0.15 ± 0.05). IPC and TAN-67 administration did not significantly increase the phosphorylation state of either isoform in the nuclear fraction throughout ischemia-reperfusion.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

We demonstrated that administration of the p38 antagonist SB-203580 attenuates cardioprotection induced by IPC; however, after administration of the ₁-opioid agonist, TAN-67, SB-203580 did not significantly attenuate cardioprotection. There was, however, a trend toward reduced cardioprotection in opioid-treated animals administered the p38 antagonist, which may suggest a minimal role for this enzyme. In fact, SB-203580 only attenuated IPC-induced cardioprotection to ~30% of the risk zone, the same extent to which TAN-67 actually induced cardioprotection. Therefore, the inability of the p38 antagonist to abolish the infarct-sparing effect of TAN-67 may be due to the reduced magnitude of opioid-induced cardioprotection versus IPC. Interestingly, when we administered the antagonist before reperfusion after sustained ischemia, cardioprotection was also attenuated, possibly via the inhibition of JNK activation that we demonstrated immediately on reperfusion.

Although it has been reported (20) that SB-203580 in the absence of IPC can induce cardioprotection (20), we did not observe any cardioprotective effect in animals administered either of two doses of SB-203580. Despite the present pharmacological data suggesting the involvement of p38 in IPC-induced cardioprotection, we were not able to detect an increase in the phosphorylation state of p38 when assessed at seven different time points throughout ischemia-reperfusion in either the cytosolic or nuclear fraction using a phosphorylation-specific primary antibody. The finding that the phosphorylation of p38 is inconsistent with cardioprotection associated with IPC and is in agreement with a previous study by Behrends and colleagues (6). These authors found that in a porcine model of ischemia-reperfusion IS reduction as a result of IPC could not be explained by an increase or decrease in p38 phosphorylation during preconditioning or during sustained ischemia.

We also demonstrated that changes in JNK phosphorylation in the nuclear fraction were not different in untreated animals versus animals administered TAN-67 or subjected to IPC. In contrast to the finding that p38 remained unchanged throughout ischemia-reperfusion, we demonstrated that the phosphorylation state of JNK is elevated in animals administered an opioid agonist or subjected to IPC in the cytosolic fraction. We demonstrated that the IPC stimulus in the absence of prolonged ischemia induced the phosphorylation of JNK, and this effect was maintained for at least the first 5 min of ischemia. In addition, we demonstrated that the phosphorylation state of both isoforms of JNK, p46 and p54, were significantly increased immediately on myocardial reperfusion versus untreated control hearts. Because a selective inhibitor of JNK in vivo is not available, these data cannot be fully interpreted. However, we suggest that the rapid increase in JNK phosphorylation on myocardial reperfusion following ischemia may be an important component of these cardioprotective interventions. In addition, the increase in JNK phosphorylation during early ischemia in preconditioned animals may account for the greater reduction in infarct size observed in these animals versus animals administered an opioid agonist.

These data concerning the activation of JNK are remarkably similar to what we have previously published concerning the role of ERK in cardioprotection. We have demonstrated that the cytosolic activation of ERK is essential for cardioprotection following either of these cardioprotective manipulations. More importantly, we provided the first evidence that opioids may induce the differential activation of ERK isoforms in cardiac muscle that may be responsible for cardioprotection. We demonstrated that, although IPC induced the activation of both p44 and p42 ERK, stimulation of the ₁-opioid receptor induced only the activation of the p44 isoform. In addition, the MEK-1 inhibitor PD-098059 blunted the in vivo cardioprotective effects of either intervention and abolished the activation of both isoforms in the cytosol. In a similar light, we again suggest that opioid-induced cardioprotection is mediated via a mechanism distinct from IPC because p38 activation is not a mediator of cardioprotection after pharmacological stimulation of the ₁-receptor but appears to be essential for cardioprotection after IPC.

The finding that p38 phosphorylation remained unchanged throughout ischemia-reperfusion and was not increased in cardioprotected animals is difficult to reconcile. However, these data are in agreement with Behrends and colleagues (6), who recently demonstrated that p38 phosphorylation during ischemia was not correlated with cardioprotection from IPC. It is known that multiple isoforms of p38 exist. Of these, p38 and - are expressed in the myocardium and they likely have opposing functions (37). It has been demonstrated that p38 is likely proapoptotic whereas p38 is antiapoptotic. Therefore, it is possible that IPC induced an increase in p38 phosphorylation while concomitantly reducing the phosphorylation of p38; hence, the assessment of p38 phosphorylation revealed no significant changes in total p38 phosphorylation. In addition, it is possible that SB-203580 is more selective for p38 than for p38; therefore, greater inhibition of p38 in vivo versus p38 may result in the abolition of any cardioprotective effects. However, this explanation seems unlikely in light of the finding that pyridinyl imidazole antagonists of p38 such as SB-203580 bind the ATP binding site and are competitive with ATP (41). Furthermore, there is no evidence that this binding site is different among - and -isoforms.

Another explanation for the present findings is that SB-203580 is not selective for inhibition of p38. Therefore, inhibition of other cardioprotective mediators by SB-203580 may account for the observed results. Indeed, Clerk and Sugden (10) have demonstrated that SB-203580 inhibited the activity of both p38 (IC₅₀ = 0.07 µM) and the 54-kDa isoform of JNK (IC₅₀ = 3-10 µM) in rat ventricular myocytes. However, they demonstrated that this compound did not affect p46 JNK activity. Furthermore, this compound has also been shown to inhibit human JNK isoforms overexpressed and immunoprecipitated from ultraviolet-treated COS-7 cells. Whitmarsh and colleagues (39) have demonstrated that SB-203580 (10 µM) selectively reduced the activity of JNK-21 and -22 (p54 JNK) to basal levels. In addition, they demonstrated that higher concentrations of SB-203580 also inhibited the activity of JNK-11 and -12 (p46 JNK). In the present study, retrospective analysis of the doses used to inhibit p38 (0.2 and 1.0 mg/kg) would likely lead to plasma concentrations of SB-203580 within the range expected to inhibit JNK activity. Therefore, because we demonstrated that JNK is activated during early ischemia and following reperfusion after IPC, inhibition of JNK in vivo by SB-203580 may explain the present results and may have contributed to much of the confusion currently surrounding the role of p38 in cardioprotection.

It has been demonstrated that a reduction in p38 activity is deleterious to cellular function and survival (24, 26), possibly via the reduction in important downstream signaling events such as an increase in MAPKAP kinase 2 activity (23) and the phosphorylation state of heat shock proteins (HSP) (2). Indeed, the effect of p38-mediated HSP phosphorylation may be important during cardioprotection (30). p38 plays a vital role in actin reorganization. Huot and colleagues (17) have demonstrated that the microfilament network of human umbilical vein epithelial cells responds to oxidant stress via a p38- and HSP27-dependent reorganization into long transcytoplasmic stress fibers. More importantly, Weinbrenner and colleagues (38) have also demonstrated in rabbit cardiomyocytes that preconditioned myocytes displayed reduced osmotic fragility, an effect that could be abolished by p38 inhibition.

Numerous reports (4, 25, 33) have also suggested that p38 activation is detrimental to cell survival. Mackay and Mochly-Rosen (21, 22) have demonstrated that prolonged p38 activation causes cell death. They have also shown that SB-203580 protected cardiac myocytes against prolonged ischemia in a dose-dependent manner, possibly via the reduced activation of caspase-3. The inhibition of this caspase likely reduces the driving force for myocyte apoptosis and in corroboration with this hypothesis, Ma and colleagues (20) have demonstrated that inhibition of p38 reduces apoptosis and improves cardiac function. In contrast to these findings, Zechner et al. (42) have demonstrated that overexpression of MKK6, which selectively activates p38 in cardiac myocytes, protected cells from apoptosis.

Few reports have investigated the importance of JNK in cardioprotection. Interestingly, Barancik and colleagues have demonstrated that, although p38 activation was detrimental to cellular viability (4), activation of the JNK signaling cascade is protective against ischemia (3). We suggest that the activation of JNK during myocardial reperfusion is important and may be attenuated by SB-203580 because this antagonist, when administered at the end of prolonged ischemia, blunted the infarct-sparing effect of IPC. Ping and colleagues (29) have demonstrated in a conscious rabbit model that activation of JNK is dependent on protein kinase C (PKC)- signaling. However, in contrast to the present investigation, they demonstrated that p46 JNK was potently activated in the nucleus. Although we did not observe p46 translocation into the nucleus, we agree with their data, which suggest that cytosolic activation of p54 is important for cardioprotection. Furthermore, it would be expected that cytosolic rather than nuclear activation of these enzymes could be important for acute cardioprotection against ischemia. However, nuclear translocation may be important for delayed cardioprotection after IPC (5, 8, 36) or ₁-opioid administration (11). Behrends and colleagues (6) demonstrated that JNK phosphorylation was not correlated with cardioprotection from IPC during early or late ischemia, although they did detect an increase in JNK phosphorylation during the reperfusion phase of IPC. However, they did not assess JNK phosphorylation during reperfusion following sustained ischemia; therefore, because we suggest that the activation of JNK during early reperfusion is important for cardioprotection, these reports are not necessarily in disagreement.

Both p38 and JNK may be activated during ischemia-reperfusion by oxidative stress. Clerk and colleagues (9) have demonstrated that H₂O₂ activated both enzymes; however, the extent of activation of p38 and subsequent activation of MAPKAP kinase 2 after H₂O₂ was comparable to that of ischemia-reperfusion but the extent of activation of JNK was less than that achieved by ischemia-reperfusion. Accordingly, the activation of p38 and MAPKAP kinase 2 could be abolished by SB-203580. Laderoute and Webster (19) also demonstrated that reactive oxygen intermediates stimulated the JNK pathway in cardiac myocytes subjected to hypoxia and subsequent reoxygenation. However, hypoxia alone was not sufficient to induce the phosphorylation of the c-jun transcription factor in their investigation. In addition, they demonstrated that this increase in kinase activity could be abolished by the tyrosine kinase inhibitor genistein. In a separate investigation, Yoshizumi et al. (40) demonstrated that H₂O₂-mediated activation of JNK was inhibited by the Src family tyrosine kinase inhibitor protein phosphatase 2. These data suggest a role for Src tyrosine kinase in the activation of JNK following oxidant stress. Interestingly, we (14) previously demonstrated that lavendustin A, a Src-endothelial growth factor receptor tyrosine kinase antagonist, could abolish the cardioprotective response to IPC. In regards to a role of JNK in apoptosis, Andreka and colleagues (1) recently demonstrated that apoptosis in cardiac myocytes induced by nitric oxide is blunted by the activation of JNK and enhanced by expression of a dominant-negative JNK, suggesting that nitric oxide activates JNK as part of a cytoprotective response.

A recent report by Saurin and colleagues (32) demonstrated that preconditioning of neonatal cultured cardiac myocytes reduces the activation of p38 during lethal ischemia and demonstrated that lethal ischemia reduced the activation of p38. This may therefore explain why we did not detect an increase in overall p38 phosphorylation during IPC. In addition, they demonstrated that expression of a dominant negative mutant of p38 protected cells from simulated ischemia. They previously demonstrated the importance of PKC- in cardioprotection because myocytes expressing constitutively active PKC- increased the resistance to simulated ischemia (43), and we have recently demonstrated that PKC- is an important component of opioid-induced cardioprotection (15). Furthermore, Saurin et al. (32) later demonstrated that overexpression of PKC- could reduce the activation of p38 during ischemia and prevent preconditioning enhancement of p38 activation following the IPC stimulus. Therefore, it is possible that PKC- activation following opioid administration protects the heart partially via the inhibition of p38 activation.

In summary, opioid-induced cardioprotection is likely mediated by a distinct mechanism from that of IPC whereby IPC utilizes the MAPK p38, whereas cardioprotection following ₁-opioid receptor stimulation is likely independent or only minimally dependent on p38 activation. The p38 antagonist SB-203580 significantly abolished cardioprotection from IPC when administered before or after the IPC stimulus but not significantly following administration of the ₁-opioid agonist TAN-67, possibly due to the lesser degree of cardioprotection obtained with TAN-67 versus IPC. However, we did not detect any changes in the phosphorylation state of p38 throughout ischemia-reperfusion in the presence of either IPC or TAN-67 treatment. We demonstrated that JNK activity was increased at certain points during ischemia-reperfusion by both interventions and suggest that SB-203580 at the doses used may be attenuating IPC via the inhibition of JNK phosphorylation.