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Am J Physiol Heart Circ Physiol 289: H785-H791, 2005. First published April 15, 2005; doi:10.1152/ajpheart.01008.2004
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Delayed adenosine A1 receptor preconditioning in rat myocardium is MAPK dependent but iNOS independent

Robert D. Lasley, Byron J. Keith, Gentian Kristo, Yukihiro Yoshimura, and Robert M. Mentzer, Jr.

Department of Surgery, University of Kentucky College of Medicine, Lexington, Kentucky

Submitted 4 October 2004 ; accepted in final form 13 April 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Adenosine A1 receptor delayed preconditioning (PC) against myocardial infarction has been well described; however, there have been limited investigations of the signaling mechanisms that mediate this phenomenon. In addition, there are multiple conflicting reports on the role of inducible nitric oxide synthase (iNOS) in mediating A1 late-phase PC. The purpose of this study was to determine the roles of the p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs) in in vivo delayed A1 receptor PC and whether this protection at the myocyte level is due to upregulation of iNOS. Myocardial infarct size was measured in open-chest anesthetized rats 24 h after treatment with vehicle or the adenosine A1 agonist 2-chloro-N6-cyclopentyladenosine (CCPA; 100 µg/kg ip). Additional rats receiving CCPA were pretreated with the p38 inhibitor SB-203580 (1 mg/kg ip) or the MAPK/ERK kinase (MEK) inhibitor PD-098059 (0.5 mg/kg ip). At 24 h after CCPA administration, a group of animals was given the iNOS inhibitor 1400W 10 min before ischemia. Treatment with CCPA reduced infarct size from 48 ± 2 to 28 ± 2% of the area at risk, an effect that was blocked by both SB-203580 and PD-098059 but not 1400W. Ventricular myocytes isolated 24 h after CCPA injection exhibited significantly reduced oxidative stress during H2O2 exposure compared with myocytes from vehicle-injected animals, and this effect was not blocked by the iNOS inhibitor 1400W. Western blot analysis of whole heart and cardiac myocyte protein samples revealed no expression of iNOS 6 or 24 h after CCPA treatment. These results indicate that adenosine A1 receptor delayed PC in rats is mediated by MAPK-dependent mechanisms, but this phenomenon is not associated with the early or late expression of iNOS.

agonist; reperfusion; infarct size; p38; extracellular signal-related kinase; cardiac myocytes; mitogen-activated protein kinase; inducible nitric oxide synthase

SIGNIFICANT EVIDENCE HAS ACCUMULATED that ischemic and pharmacological preconditioning (PC) against myocardial ischemia-reperfusion injury occurs in two distinct phases. The effects of acute PC may persist for ≤60 min, after which protection is lost. A second window of protection, also referred to as delayed or late-phase PC, appears 24 h after the PC stimulus and may persist for up to 72 h (5, 6, 29). The primary difference between acute and delayed PC appears to be the role of induced or increased expression of various proteins [such as inducible NO synthase (iNOS), manganese superoxide dismutase, and cyclooxygenase-2, to name a few] after the acute PC stimulus (5, 6, 9, 18, 32, 41).

Despite numerous reports implicating the upregulation of various cardioprotective proteins in delayed PC, there have been few reports investigating the upstream signaling pathways in this phenomenon. The stimulation of one or more mitogen-activated protein kinases (MAPKs) such as the p38 and p42/p44 extracellular signal-related kinase (ERK) MAPK families results in the translocation of these active kinases to the nucleus to initiate gene transcription (24). In the only in vivo study to date addressing this signaling mechanism, Fryer et al. (11) reported that opioid-induced late-phase PC was blocked by both p38 and ERK inhibitors. Although there are numerous reports of adenosine A1 receptor delayed PC (2–4, 7–10, 21–23, 26, 38, 46, 47) and evidence that adenosine and A1 receptor agonists induce the activation of both p38 and ERK (1, 13, 15, 25, 34, 35, 37), there have been no in vivo studies determining the role of these kinases in delayed PC.

Another unresolved aspect of A1 receptor delayed PC is what specific proteins are upregulated in this phenomenon. The protein that has received the most attention in mediating delayed ischemic and pharmacological PC is iNOS. Although adenosine A1 receptor agonists induce delayed PC in multiple species, there is conflicting evidence for the involvement of iNOS (4, 8, 30, 31, 47). Two different iNOS inhibitors failed to block A1 delayed PC in rabbit myocardium (8). Two studies in murine myocardium utilizing iNOS-knockout mice have also yielded conflicting results on the role of iNOS in adenosine A1 receptor late-phase PC (4, 47). In addition, although there is evidence that delayed ischemic PC in murine and canine myocardium is associated with iNOS upregulation in cardiac myocytes (19, 41), there have been no reports of direct testing of whether A1 delayed PC protects cardiac myocytes via an iNOS-dependent mechanism.

The present study was designed to address these limitations in the literature. In vivo studies were conducted to determine whether A1 receptor delayed PC against myocardial infarction in rats is dependent on p38 and/or ERK MAPKs. The role of iNOS in mediating this phenomenon was tested both in adult rat ventricular myocytes submitted to oxidative stress and in in vivo rat hearts.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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All animals in this study received humane care according to the guidelines set forth in the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, 1996) and according to the guidelines of the University of Kentucky Institutional Animal Care and Use Committee.

In vivo regional ischemia preparation. Male Sprague-Dawley rats (300–375 g body wt) were anesthetized with ketamine (75 mg/kg ip) and xylazine (1 mg/kg ip). Anesthesia was maintained with additional ketamine and xylazine as needed. A tracheostomy was performed, and the rats were ventilated (model 683; Harvard Apparatus; Natick, MA) with a mixture of room air and 100% O2. Tidal volume, respiratory rate, and percent O2 in the inspired air were adjusted to maintain normal arterial blood gas levels and pH values. The right femoral artery was cannulated with a 24-gauge catheter for blood pressure measurements and blood gas analysis. The left jugular vein was cannulated for the infusion of fluids and drugs. Body temperature was monitored with a rectal temperature probe and maintained at 37.5°C with a heating pad.

The heart was exposed by midline sternotomy, and the pericardium was removed. A ligature (7-0; Prolene) was passed below the left coronary artery ~4–5 mm from its origin. The ends of the suture were threaded through a small piece of soft vinyl tube to form a snare. Ischemia was induced by gently pulling the snare and then fixing it by clamping the tube. Epicardial cyanosis and a subsequent decrease in blood pressure verified coronary artery occlusion. Reperfusion was initiated by unclamping the snare and was confirmed by visible epicardial hyperemia. Animals were allowed a period of 30 min after all instrumentation before experimental protocols were initiated.

In vivo experimental protocols. Figure 1 illustrates the experimental protocol for the in vivo infarct protocols. All rats were subjected to 25 min of regional ischemia and 2 h of reperfusion. Control rats were administered vehicle (10% DMSO in saline; 1 ml ip; n = 10 rats) 24 h before the open-chest ischemia protocol. Adenosine A1 receptor delayed PC was induced by injecting rats with the A1 agonist 2-chloro-N6-cyclopentyladenosine (CCPA; 100 µg/kg ip; n = 9 rats) 24 h before the experiment. Additional rats were treated 30 min before CCPA injection with the p38 inhibitor SB-203580 (1 mg/kg ip; n = 6 rats) or the MEK inhibitor PD-098059 (0.5 mg/kg ip; n = 4 rats). At 24 h after CCPA administration, a group of animals was given the iNOS inhibitor 1400W (1 mg/kg iv; n = 5 rats) 10 min before the coronary artery occlusion. An additional group of rats was treated with the adenosine A2a agonist CGS-21680 (10 or 100 µg/kg ip; n = 4 rats/group) 24 h before ischemia. CCPA, PD-098059, SB-203580, 1400W, and CGS-21680 were made as concentrated stock solutions in DMSO and were diluted in saline immediately before use.



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  		Fig. 1. Protocols for the in vivo myocardial infarction experiments. Rats were administered intraperitoneal injections of vehicle, adenosine agonists, and MAPK inhibitors 24 h before the open-chest protocols. The p38 and mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors, SB-203580 (SB) and PD-098059 (PD), respectively, were given 30 min before the 2-chloro-N6-cyclopentyladenosine (CCPA) injection. Inducible nitric oxide synthase (iNOS) inhibitor 1400W was administered 10 min before ischemia. CGS-21680 (CGS), is an adenosine A2a agonist.

 
Determination of infarct size. After 2 h of reperfusion, the coronary artery was reoccluded, and 1.5 ml of 5% Evans blue solution was injected via the jugular vein. The ischemic area at risk (AAR) was devoid of the Evans blue stain. The heart was excised, and the left ventricle (LV) was removed from the remaining tissue and subsequently cut into four slices of equal thickness in a plane parallel to the atrioventricular groove. The slices were incubated in a 1.0% triphenyltetrazolium chloride (TTC) solution in phosphate-buffered saline solution at 37°C for 15 min. Slices were then compressed to a uniform thickness of 2 mm by placing them between two transparent Plexiglas plates. The area of nonischemic LV (stained blue), the AAR (stained red by TTC), and the infarcted area (TTC negative) were quantified using graphic analysis software (SigmaScan Pro automated image-analysis software; Jandel Scientific; SPSS; San Rafael, CA). AAR was expressed as a percentage of the LV (AAR/LV), and infarct size (IS) was expressed as a percentage of the AAR (IS/AAR).

Isolation of rat ventricular myocytes. Ventricular myocytes were isolated from adult male Sprague-Dawley rats (275–325 g body wt) as previously described (21) with minor modifications. The excised heart was perfused with HEPES medium that contained (in mM) 118 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 10 HEPES, 11 glucose, and 2 pyruvate, pH 7.2 for 5 min in a nonrecirculating mode. Collagenase (Liberase type 2; 0.14 mg/ml; Roche Biochemicals; Indianapolis, IN) was then added to the medium and recirculated for 15–20 min. During the final 10 min, calcium was gradually reintroduced to a final concentration of 500 µM. The heart was removed from the cannula and minced, and the myocytes were mechanically dispersed. Dissociated myocytes were suspended in HEPES buffer (pH 7.40, 1 mM CaCl2) until use. Myocytes were allowed to equilibrate for 60 min before protocols were initiated. Experiments were conducted in HEPES buffer within 6 h of isolation. Rats were injected with vehicle or CCPA (100 µg/kg ip) 24 h before isolation of ventricular myocytes.

Isolated myocyte protocols. Myocytes were exposed to H2O2, and the extent of oxidative stress was determined with the fluorescent dye dichlorofluorescein diacetate (DCFDA) as previously described (21). Myocytes were loaded with DCFDA (5 µM) for 15 min at room temperature in the dark. The myocytes were washed with HEPES medium to remove extracellular dye and incubated for an additional 30 min to allow for intracellular deacetylation of DCFDA, which results in the formation of the reduced and weakly fluorescent dichlorofluorescein moeity. Upon oxidation by reactive oxygen and reactive nitrogen species (ROS and RNS, respectively), reduced dichlorofluorescin is converted to the highly fluorescent dichlorofluorescein (DCF).

An aliquot of dye-loaded myocytes was then allowed to settle on laminin-coated coverslips placed in a 300-µl temperature-controlled recording chamber (model RC-24 chamber and model TC-324B temperature controller; Warner Instruments; Hamden, CT) on the stage of an Olympus IX-70 inverted microscope (Olympus America; Melville, NY). Cells were suffused with normal HEPES buffer (pH 7.4 at 37°C) at a flow rate of 1 ml/min. Two baseline DCF fluorescence values were recorded after 5 and 20 min of suffusion before the cells were exposed to H2O2 (150 µM), which induced oxidative stress that was maintained for 10 min. All cell fluorescence values were background corrected, and increases in DCF during H2O2 exposure were expressed relative to each cell's baseline fluorescence.

An excitation wavelength of 490 nm was used, and emission fluorescence was collected at 520 ± 5 nm. To reduce excitation-dependent oxidation of the dye, a neutral-density filter (ND12) was placed in the path of the excitation source (a 75-W xenon arc lamp), and sampling times were limited to 160 ms. Epifluorescence was collected by a charge-coupled device camera attached to a side port of the microscope, and fluorescence intensity analysis was done on a Pentium processor with custom-made software.

A subset of CCPA-preconditioned myocytes was treated with the selective iNOS inhibitor 1400W (1 µM). Treatment was initiated for 15 min before the final baseline DCF measurement was made and was continued throughout the H2O2 exposure.

Western blot analysis. To determine whether A1 receptor delayed-phase PC was associated with upregulation of iNOS, rats (n = 3) were injected with vehicle or CCPA (100 µg/kg ip) 6 and 24 h before isolation of ventricular myocytes. Myocytes were washed with ice-cold PBS and resuspended in two volumes of ice-cold buffer A that contained 250 mM sucrose, 20 mM HEPES, 10 mM KCl, and 1.5 mM MgCl2, pH 7.4 with aprotinin (2 µg/ml), leupeptin (2 µg/ml), pepstatin A (2 µg/ml), benzamidine (0.1 mM), and phenylmethylsulfonyl fluoride (0.1 mM). This suspension received 20 strokes with a Teflon Dounce homogenizer on ice, which was followed by sonication (three times for 10 s each at 4°C) and centrifugation (750 g, for 10 min at 4°C) to sediment the nuclear fraction. The postnuclear supernatant was centrifuged at 105,000 g for 1 h to generate cytosolic fractions. Additional rats received injections of lipopolysaccharide (LPS, 10 mg/kg ip) either 6 or 16 h before isolation of the heart. Whole heart and liver cytosolic fractions were generated as described above after hearts were homogenized with a Polytron. Protein concentrations were determined by Bradford assay.

Proteins (50–120 µg) from the myocyte-, heart-, and liver-soluble fractions were subjected to 8% SDS-PAGE under reducing conditions. After electrophoresis, the proteins were electroblotted to polyvinylidene difluoride membranes. The membranes were blocked (for 1 h) with Tris-buffered saline (TBS) that contained 5% milk and 0.5% Tween 20. Incubation (overnight at 4°C) of anti-iNOS (1:1,000 dilution) affinity-purified IgG (Transduction Labs; Lexington, KY) in TBS (1% milk with 0.2% Tween 20) was followed by washes with TBS (0.2% milk with 0.2% Tween 20). Secondary antibody (1:7,500 dilution) conjugated with horseradish peroxidase was incubated (for 1 h) with TBS (1% milk with 0.2% Tween 20) followed by washing. The membrane was placed in enhanced chemiluminescent substrate (for 5 min) and exposed to Kodak X-OMAT film for ≤10 min.

Chemicals. PD-098059 and SB-203580 were purchased from LC Laboratories (Woburn, MA). CCPA, CGS-21680, 1400W, and LPS were obtained from Sigma-Aldrich (St. Louis, MO). DCFDA was purchased from Molecular Probes (Eugene, OR).

Data analysis. Data are expressed as means ± SE. Among-group differences in infarct size were determined by one-way ANOVA. For the H2O2 protocols, 6–10 myocytes were studied from 4 or 5 myocyte isolations. Group differences were determined by a two-way ANOVA and subsequent Newman-Keuls post hoc analysis. A P value < 0.05 was considered statistically significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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In vivo results. Seven animals were excluded from the study, three due to technical errors and four due to a mean blood pressure < 30 mmHg during reperfusion. A summary of the hemodynamic parameters during the ischemia-reperfusion protocols is shown in Table 1. There were no differences among the groups in heart rate or mean arterial pressure (MAP) before or during ischemia or during reperfusion. Coronary occlusion was associated with decreased MAP (vs. baseline values) in the vehicle control, 24-h CCPA, PD-098059 with CCPA, CCPA with 1400W, and high-dose CGS-21680 groups, which persisted throughout reperfusion in the former four groups.


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  		Table 1. Hemodynamics during in vivo regional ischemia protocol

 
Figure 2 summarizes the in vivo IS findings. There were no significant differences in the sizes of the ischemic zones, which were ~35% of the LV. Rats injected with vehicle 24 h before the open-chest protocol exhibited an IS of 48 ± 2% of the AAR (Fig. 2). IS after CCPA 24-h PC was 28 ± 2%, which is a 42% reduction. Treatments with the p38 and MEK inhibitors SB-203580 and PD-098059, respectively, before CCPA injection resulted in ISs of 40 ± 2 and 50 ± 4%, respectively. Preischemic administration of the iNOS inhibitor 1400W in rats injected with CCPA 24 h before the experiment was associated with 31 ± 1% infarction. Treatment with two doses of the adenosine A2a agonist CGS-21680 24 h before regional ischemia had no effect on IS (42 ± 5 and 41 ± 7% in the low- and high-dose groups, respectively).



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  		Fig. 2. In vivo infarct size results. Infarct size was determined by triphenyltetrazolium chloride staining after 25 min of coronary artery occlusion and 2 h of reperfusion. Results are expressed as a percentage of the left ventricular area at risk (AAR). *P < 0.05 vs. vehicle-injected rats.

 
In vitro results. In cells loaded with the reactive species-sensitive fluorescent indicator DCFDA, exposure to H2O2 (150 µM) resulted in a significant increase in DCF fluorescence. As shown in Fig. 3 in myocytes (n = 43) isolated from saline-injected rats, DCF fluorescence increased 10.7 ± 2.3- and 33.4 ± 4.3-fold compared with baseline values after 5 and 10 min of exposure to H2O2. In contrast, in ventricular myocytes (n = 44) from CCPA-injected rats, DCF fluorescence increased only 5.6 ± 0.7-fold after 5 min of H2O2 exposure and 15.4 ± 1.2-fold after 10 min of H2O2 exposure (both values, P < 0.05 vs. myocytes from vehicle-injected rats). Results in Fig. 2 also indicate that CCPA 24-h PC of cardiac myocytes was not blocked by the iNOS inhibitor 1400W, as myocytes in this group (n = 29) exhibited identical increases in DCF fluorescence as those in the absence of this inhibitor.



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  		Fig. 3. Dichlorofluorescein (DCF) flourescence in isolated ventricular myocytes exposed to H2O2 (150 µM). Myocytes were isolated from vehicle or CCPA-injected rats 24 h after treatment. DCF fluorescence for each cell was normalized to baseline values. The iNOS inhibitor 1400W was applied to the myocytes for 15 min before H2O2 exposure. *P < 0.05 vs. vehicle-injected myocytes. PC, preconditioned.

 
The Western blot results were consistent with the above pharmacological observations. Figure 4 shows that iNOS immunoreactivity was not observed in myocyte cytosolic fractions (lanes 2–4) isolated from vehicle-injected rats or in normal liver cytosol (lane 8). Cytosolic fractions (lanes 5 and 6) obtained from myocytes isolated from CCPA-injected rats showed no expression of iNOS, similar to what was observed in the vehicle-injected animals. Protein from LPS-injected rats was analyzed as a positive control. Robust expression of iNOS was observed in whole heart (lane 1) and liver (lane 7) cytosolic fractions 6 h after LPS treatment, although to a much greater extent in the liver.



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  		Fig. 4. Western blot analysis of myocyte cytosolic fractions 24 h after treatment. Myocyte cytosolic proteins (50 µg) from vehicle- (lanes 2–4) and CCPA-injected (lanes 5 and 6) rats were loaded onto the gel. Heart (lane 1) and liver (lane 7) cytosolic fractions (50 µg) from rats treated with lipopolysaccharide (LPS; 6 h prior) were used as iNOS-positive controls. Lane 8 is the vehicle-treated liver cytosolic fraction. MW, Molecular weight.

 
In the course of generating the LPS-positive controls, we observed that there was much greater expression of whole heart iNOS at 6 h (lane 7) after LPS treatment than after 16 h (lane 8; Fig. 5). To exclude the possibility that iNOS was induced only in the early phase of delayed A1 agonist PC, protein was obtained from myocytes 6 h after CCPA treatment. Cardiac myocytes from rats isolated 6 h after CCPA treatment (lanes 3 and 4) exhibited no inducement of iNOS, similar to results observed after 24 h. Examination of endothelial NOS (eNOS) expression revealed no difference between CCPA- and saline-treated animals (data not shown).



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  		Fig. 5. Western blot analysis of iNOS after 6 h of treatment. Lanes 1 and 2 are liver protein samples (20 µg) obtained 6 h after LPS (lane 1) or saline (lane 2) treatment, respectively. Lanes 3 and 4 are myocyte cytosol (50 µg) obtained 6 h after CCPA injection. Lanes 5 and 6 are CCPA and saline 24-h samples, respectively. Lanes 7 and 8 show supernatant from whole heart (50 µg) after 6 or 16 h of LPS treatment, respectively.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The results of this study indicate that A1 receptor delayed PC against infarction in rat myocardium appears to be mediated by the activation of both p38 and ERK MAPKs. Delayed A1 PC was evident at the cardiac myocyte level, as ventricular myocytes isolated from A1 agonist-injected rats exhibited increased resistance to oxidative stress. These beneficial effects, however, were not blocked by the iNOS inhibitor 1400W, nor were they associated with the inducement of iNOS in either the whole heart or cardiac myocytes.

Ischemic and pharmacological PC have been shown to exert both acute and delayed protection against myocardial cell death in multiple preparations and in every species tested to date including human cardiac tissue (5–7, 22, 26, 29). The observations that acute PC wanes after several hours but then reappears 24 h later has led to the hypothesis that this phenomenon is due to the upregulation or inducement of one or more cardioprotective proteins such as antioxidant enzymes, iNOS, and cyclooxygenase-2 (5, 6, 9, 18, 32, 41). One mechanism for increasing gene expression is the activation of MAPKs such as the p38 and p42/p44 ERK. The activation of MAPKs by extracellular stimuli and G protein-receptor agonists results in the phosphorylation of numerous substrates including various transcription factors that regulate gene expression (24).

There have been several reports to date implicating the p38 pathway in acute and delayed ischemic and pharmacological PC (11, 12, 27). There is also evidence that adenosine receptor activation can activate p38 MAPK in several tissues including heart (13, 15, 25, 35, 37). We (1) recently reported that acute adenosine receptor PC is associated with increased p38 activation before ischemia. Despite these findings, there have been few reports of the role of p38 in adenosine A1 receptor delayed PC. In the only studies to date, the p38 inhibitor SB-203580 has been reported to block A1 agonist late-phase PC in isolated mouse heart (46) and in human atrial tissue (7, 26). The results of an additional study (10) indicated that rabbit myocardium exhibited a significant increase in p38 activity 24 h after A1 agonist treatment, but the effects of p38 inhibition were not assessed. Fryer et al. (11) reported that the p38 inhibitor SB-203580 blocked opioid receptor late-phase PC in in vivo rat myocardium. The results of the present study suggest that p38 plays a key role in mediating in vivo adenosine A1 receptor delayed PC in rat myocardium.

In contrast to the several reports of p38 involvement in late-phase PC, there has been only one study published to date on the role of ERK in this phenomenon. Fryer et al. (11) reported that the same MEK inhibitor that we used in the present study, PD-098059, blocked {delta}-opioid agonist delayed PC in in vivo rat myocardium. Adenosine receptor activation, including the A1 receptor, activates ERK in several tissues including neonatal rat cardiac myocytes (13, 15, 35, 37). We recently reported that acute adenosine receptor PC in in vivo rat myocardium is associated with significant activation of both p42 and p44 isoforms (34). Our findings are the first report that the MEK inhibitor PD-098059 blocks A1 receptor delayed PC, which suggests a key role for the downstream ERK pathway in mediating this phenomenon.

Although there have been numerous reports of adenosine A1 delayed PC, there has been only one study determining the effects of A2a receptor agonists. Takano et al. (7) reported that the A2a receptor agonist CGS-21680 did not induce delayed PC against myocardial infarction in rabbits. We observed similar results in the present study with two different doses of CGS-21680. On the surface, these results may be expected; however, there are reports that A2a receptor stimulation, in several tissues including the heart, activates MAPK ERK (13, 20, 37, 43). Studies of A2a receptor modulation of ERK in intact myocardium, however, have been limited to reduction of infarct size via reperfusion A2a agonist treatments (20, 43). Thus cardiac A2a receptors may not couple to the ERK pathway in normal myocardium.

Because activated p38 and ERK translocate to the nucleus to phosphorylate transcription factors, the evidence that p38 and ERK MAPKs are components of delayed PC is consistent with the hypothesis that this phenomenon is due to the upregulation of protective proteins. One of the proteins that has received the most attention is iNOS. Although there are multiple reports showing that both ischemic and pharmacological delayed PC are associated with the upregulation of iNOS (14, 18, 19, 32, 40–42, 44, 45, 47), there are conflicting reports on whether this protein plays a role in adenosine A1 receptor late-phase PC (4, 8, 30, 31, 47). Takano et al. (38) first reported that adenosine A1 delayed PC occurred via an NOS-dependent pathway in rabbit myocardium, although it was not determined whether iNOS was the mediator. In a subsequent study, also in rabbit myocardium, two different iNOS inhibitors, both of which blocked delayed ischemic PC, failed to inhibit A1 receptor delayed PC (8).

There are similar conflicting reports for murine myocardium. One report indicated that the A1 receptor agonist CCPA increased iNOS protein expression in wild-type mice, and delayed A1 PC in isolated perfused mouse hearts was blocked by an iNOS inhibitor (47). In addition, CCPA did not induce late-phase PC in iNOS-knockout mice (47). In contrast, Bell and colleagues (4) reported that, although A1 agonist treatment did increase iNOS upregulation in wild-type mice, this did not appear to be necessary for delayed PC, because delayed A1 PC could be still be induced in iNOS-knockout mice. These authors in fact observed that eNOS was upregulated 24 h after CCPA treatment (4). Nayeem et al. (30) reported that isolated cardiac myocytes from A1 receptor-overexpressing mice did require iNOS for protection against simulated ischemia-induced cell death but that adenosine A1 delayed PC could be induced in myocytes from iNOS-knockout mice (31). However, both of these conclusions were based solely on results obtained with iNOS inhibitors, and iNOS expression was not examined.

The results of the present study indicate that adenosine A1 receptor delayed PC in rat myocardium is not associated with the expression of iNOS in ventricular myocytes. These results are consistent with the inability of the selective iNOS inhibitor 1400W to inhibit the anti-infarct effects of 24 h of PC with CCPA or to block the increased resistance to oxidative stress in cardiomyocytes isolated from CCPA-injected rats. Our findings are consistent with those of Igarashi et al. (16), who reported that iNOS induction in neonatal rat cardiomyocytes was associated with exacerbation of injury during oxidative stress. In addition, adenosine A1 receptor agonist pretreatment has been shown to decrease iNOS upregulation and mortality in mice after LPS treatment (28). Treatment with LPS was associated with robust myocardial iNOS expression 6 h after treatment, which was significantly diminished after 16 h. This same transient pattern of iNOS expression has been reported after LPS treatment in mice (28) and during monophosphoryl lipid A-induced PC in rats (40) and pigs (44). Given these time-dependent observations, we tested for myocyte iNOS expression 6 h after CCPA treatment, which also turned up negative. We were also not able to detect any iNOS expression in the whole heart. These findings suggest that iNOS is neither a trigger nor a mediator of adenosine A1 receptor delayed PC in rat myocardium.

Although numerous investigators have reported the upregulation of iNOS in late-phase ischemic and pharmacological PC (14, 18, 19, 32, 40–42, 44, 45, 47), there have been few attempts to determine the cellular or subcellular source of this protein. The importance of this issue is supported by the findings of Poon et al. (33), who reported that the differential expression of iNOS in myocytes and neutrophils during sepsis determined whether this protein exerted beneficial or detrimental effects. Late-phase ischemic PC in mouse myocardium is associated with increased iNOS expression with some immunocytochemical evidence that this occurred in myocytes (41). However, there was substantial constitutive iNOS expression in both cytosolic and membrane fractions, and iNOS expression increased in the nonischemic zones. In contrast, we observed essentially no iNOS in normal rat myocardium or myocytes. Ventricular myocytes isolated from canine myocardium 24 h after ischemic PC did exhibit functional evidence of increased iNOS expression, but immunohistochemical analysis of ventricular myocardium revealed only sparse myocyte iNOS with the greatest iNOS expression in the perivascular space (19). Rui et al. (36) observed that delayed PC (using anoxia-reoxygenation) could be induced in neonatal cardiomyocytes isolated from both wild-type and iNOS-deficient mice, and delayed PC in the former was not associated with upregulation of iNOS. To our knowledge the present findings are the first to directly examine whether delayed pharmacological PC is associated with the inducement or upregulation of a specific protein in isolated ventricular myocytes. Our functional and biochemical findings indicate no role for cardiomyocyte iNOS in mediating adenosine A1 receptor delayed PC in rat myocardium.

In summary, in vivo adenosine A1 receptor delayed PC against myocardial infarction is mediated by both p38 and ERK-dependent pathways. This protection extends to the cardiomyocyte level, as isolated ventricular myocytes from A1 agonist-injected rats exhibited significantly reduced oxidative stress during H2O2 exposure. However, A1 receptor delayed PC in rat myocardium is not associated with the inducement of iNOS.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the National Heart, Lung, and Blood Institute Grants HL-34579 (to R. M. Mentzer, Jr.) and HL-66132 (to R. D. Lasley).


   ACKNOWLEDGMENTS
 
The authors thank Drs. Eric L. Kilpatrick and Prakash Narayan for assistance in these studies.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. D. Lasley, Dept. of Surgery, Univ. of Kentucky College of Medicine, MN276, Chandler Medical Center, 800 Rose St., Lexington, KY 40536-0298 (E-mail: rlasley{at}uky.edu)

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. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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  1. Ballard-Croft C, Kristo G, Yoshimura Y, Reid E, Keith BJ, Mentzer RM Jr, and Lasley RD. Acute adenosine preconditioning is mediated by p38 MAPK activation in discrete subcellular compartments. Am J Physiol Heart Circ Physiol 288: H1359–H1366, 2005.[Abstract/Free Full Text]
  2. Baxter GF, Marber MS, Patel VC, and Yellon DM. Adenosine receptor involvement in a delayed phase of myocardial protection 24 hours after ischemic preconditioning. Circulation 90: 2993–3000, 1994.[Abstract/Free Full Text]
  3. Baxter GF and Yellon DM. Time course of delayed myocardial protection after transient adenosine A1-receptor activation in the rabbit. J Cardiovasc Pharmacol 29: 631–638, 1997.[CrossRef][Web of Science][Medline]
  4. Bell RM, Smith CC, and Yellon DM. Nitric oxide as a mediator of delayed pharmacological (A1 receptor triggered) preconditioning; is eNOS masquerading as iNOS? Cardiovasc Res 53: 405–413, 2002.[Abstract/Free Full Text]
  5. Bolli R. The late phase of preconditioning. Circ Res 87: 972–983, 2000.[Abstract/Free Full Text]
  6. Bolli R. Cardioprotective function of inducible nitric oxide synthase and role of nitric oxide in myocardial ischemia and preconditioning: an overview of a decade of research. J Mol Cell Cardiol 33: 1897–1918, 2001.[CrossRef][Web of Science][Medline]
  7. Carroll R and Yellon DM. Delayed cardioprotection in a human cardiomyocyte-derived cell line: the role of adenosine, p38MAP kinase and mitochondrial KATP. Basic Res Cardiol 95: 243–249, 2000.[CrossRef][Web of Science][Medline]
  8. Dana A, Baxter GF, and Yellon DM. Delayed or second window preconditioning induced by adenosine A1 receptor activation is independent of early generation of nitric oxide or late induction of inducible nitric oxide synthase. J Cardiovasc Pharmacol 38: 278–287, 2001.[CrossRef][Web of Science][Medline]
  9. Dana A, Jonassen AK, Yamashita N, and Yellon DM. Adenosine A1 receptor activation induces delayed preconditioning in rats mediated by manganese superoxide dismutase. Circulation 101: 2841–2848, 2000.[Abstract/Free Full Text]
  10. Dana A, Skarli M, Papakrivopoulou J, and Yellon DM. Adenosine A1 receptor induced delayed preconditioning in rabbits: induction of p38 mitogen-activated protein kinase activation and Hsp27 phosphorylation via a tyrosine kinase- and protein kinase C-dependent mechanism. Circ Res 86: 989–997, 2000.[Abstract/Free Full Text]
  11. Fryer RM, Hsu AK, and Gross GJ. ERK and p38 MAP kinase activation are components of opioid-induced delayed cardioprotection. Basic Res Cardiol 96: 136–142, 2001.[CrossRef][Web of Science][Medline]
  12. Fryer RM, Pratt PF, Hsu AK, and Gross GJ. Differential activation of extracellular signal regulated kinase isoforms in preconditioning and opioid-induced cardioprotection. J Pharmacol Exp Ther 296: 642–649, 2001.[Abstract/Free Full Text]
  13. Germack R and Dickenson JM. Characterization of ERK1/2 signalling pathways induced by adenosine receptor subtypes in newborn rat cardiomyocytes. Br J Pharmacol 141: 329–339, 2004.[CrossRef][Web of Science][Medline]
  14. Guo Y, Jones WK, Xuan YT, Tang XL, Bao W, Wu WJ, Han H, Laubach VE, Ping P, Yang Z, Qiu Y, and Bolli R. The late phase of ischemic preconditioning is abrogated by targeted disruption of the inducible NO synthase gene. Proc Natl Acad Sci USA 96: 11507–11512, 1999.[Abstract/Free Full Text]
  15. Haq SEA, Clerk A, and Sugden PH. Activation of mitogen-activated protein kinases (p38-MAPKs, SAPKs/JNKs and ERKs) by adenosine in the perfused rat heart. FEBS Lett 434: 305–308, 1998.[CrossRef][Web of Science][Medline]
  16. Igarashi J, Nishida M, Hoshida S, Yamashita N, Kosaka H, Hori M, Kuzuya T, and Tada M. Inducible nitric oxide synthase augments injury elicited by oxidative stress in rat cardiac myocytes. Am J Physiol Cell Physiol 274: C245–C252, 1998.[Abstract/Free Full Text]
  17. Imagawa J, Yellon DM, and Baxter GF. Pharmacological evidence that inducible nitric oxide synthase is a mediator of delayed preconditioning. Br J Pharmacol 126: 701–708, 1999.[CrossRef][Web of Science][Medline]
  18. Jones WK, Flaherty MP, Tang XL, Takano H, Qiu Y, Banerjee S, Smith T, and Bolli R. Ischemic preconditioning increases iNOS transcript levels in conscious rabbits via a nitric oxide-dependent mechanism. J Mol Cell Cardiol 31: 1469–1481, 1999.[CrossRef][Web of Science][Medline]
  19. Kim SJ, Kim YK, Takagi G, Huang CH, Geng YJ, and Vatner SF. Enhanced iNOS function in myocytes one day after brief ischemic episode. Am J Physiol Heart Circ Physiol 282: H423–H428, 2002.[Abstract/Free Full Text]
  20. Kis A, Baxter GF, and Yellon DM. Limitation of myocardial reperfusion injury by AMP579, an adenosine A1/A2A receptor agonist: role of A2A receptor and Erk1/2. Cardiovasc Drugs Ther 17: 415–425, 2003.[CrossRef][Web of Science][Medline]
  21. Kodani E, Shinmura K, Xuan YT, Takano H, Auchampach JA, Tang XL, and Bolli R. Cyclooxygenase-2 does not mediate late preconditioning induced by activation of adenosine A1 or A3 receptors. Am J Physiol Heart Circ Physiol 281: H959–H968, 2001.[Abstract/Free Full Text]
  22. Kristo G, Yoshimura Y, Keith BJ, Stevens RM, Jahania SA, Mentzer RM Jr, and Lasley RD. The adenosine A1/A2A receptor agonist AMP579 induces both acute and delayed preconditioning against in vivo myocardial stunning. Am J Physiol Heart Circ Physiol 287: H2746–H2753, 2004.[Abstract/Free Full Text]
  23. Kudo M, Wang Y, Xu M, Ayub A, and Ashraf M. Adenosine A1 receptor mediates late preconditioning via activation of PKC-delta signaling pathway. Am J Physiol Heart Circ Physiol 283: H296–H301, 2002.[Abstract/Free Full Text]
  24. Luttrell DK and Luttrell LM. Signaling in time and space: G protein-coupled receptors and mitogen-activated protein kinases. Assay Drug Dev Technol 1: 327–338, 2003.[CrossRef][Web of Science][Medline]
  25. Liu Q and Hofmann PA. Modulation of protein phosphatase 2a by adenosine A1 receptors in cardiomyocytes: role for p38 MAPK. Am J Physiol Heart Circ Physiol 285: H97–H103, 2003.[Abstract/Free Full Text]
  26. Loubani M and Galinanes M. Pharmacological and ischemic preconditioning of the human myocardium: mitoKATP channels are upstream and p38MAPK is downstream of PKC. BMC Physiol 2: 10, 2002.[CrossRef][Medline]
  27. Michel MC, Li Y, and Heusch G. Mitogen-activated protein kinases in the heart. Naunyn Schmiedebergs Arch Pharmacol 363: 245–266, 2001.[CrossRef][Web of Science][Medline]
  28. Moochhala SM, Hon WM, Chhatwal VJ, and Khoo HE. Expression of inducible nitric oxide synthase in mice: pharmacological evaluation of adenosine receptor agonists. Eur J Pharmacol 316: 287–296, 1996.[Medline]
  29. Mubagwa K and Flameng W. Adenosine, adenosine receptors and myocardial protection: an updated overview. Cardiovasc Res 52: 25–39, 2001.[Abstract/Free Full Text]
  30. Nayeem MA, Matherne GP, and Mustafa SJ. Ischemic and pharmacological preconditioning induces further delayed protection in transgenic mouse cardiac myocytes over-expressing adenosine A1 receptors (A1AR): role of A1AR, iNOS and KATP channels. Naunyn Schmiedebergs Arch Pharmacol 367: 219–226, 2003.[CrossRef][Web of Science][Medline]
  31. Nayeem MA and Mustafa SJ. Ischemic and pharmacological induction of delayed cellular protection in iNOS gene-disrupted mice myocytes. Pol J Pharmacol 55: 73–79, 2003.[Medline]
  32. Patel HH, Hsu AK, and Gross GJ. COX-2 and iNOS in opioid-induced delayed cardioprotection in the intact rat. Life Sci 75: 129–140, 2004.[CrossRef][Web of Science][Medline]
  33. Poon BY, Raharjo E, Patel KD, Tavener S, and Kubes P. Complexity of inducible nitric oxide synthase: cellular source determines benefit versus toxicity. Circulation 108: 1107–1112, 2003.[Abstract/Free Full Text]
  34. Reid EA, Kristo G, Yoshimura Y, Ballard-Croft C, Keith BJ, Mentzer RM Jr, and Lasley RD. In vivo adenosine receptor preconditioning reduces myocardial infarct size via subcellular ERK signaling. Am J Physiol Heart Circ Physiol 288: H2253–H2259, 2005.[Abstract/Free Full Text]
  35. Robinson AJ and Dickenson JM. Regulation of p42/p44 MAPK and p38 MAPK by the adenosine A1 receptor in DDT1MF-2 cells. Eur J Pharmacol 413: 151–161, 2001.[CrossRef][Web of Science][Medline]
  36. Rui T, Cepinskas G, Feng Q, and Kvietys PR. Delayed preconditioning in cardiac myocytes with respect to development of a proinflammatory phenotype: role of SOD and NOS. Cardiovasc Res 59: 901–911, 2003.[Abstract/Free Full Text]
  37. Schulte G and Fredholm BB. Signalling from adenosine receptors to mitogen-activated protein kinases. Cell Signal 15: 813–827, 2003.[CrossRef][Web of Science][Medline]
  38. Takano H, Bolli R, Black RG Jr, Kodani E, Tang XL, Yang Z, Bhattacharya S, and Auchampach JA. A1 or A3 adenosine receptors induce late preconditioning against infarction in conscious rabbits by different mechanisms. Circ Res 88: 520–528, 2001.[Abstract/Free Full Text]
  39. Takano H, Manchikalapudi S, Tang XL, Qiu Y, Rizvi A, Jadoon AK, Zhang Q, and Bolli R. Nitric oxide synthase is the mediator of late preconditioning against myocardial infarction in conscious rabbits. Circulation 98: 441–449, 1998.[Abstract/Free Full Text]
  40. Tosaki A, Maulik N, Elliott GT, Blasig IE, Engelman RM, and Das DK. Preconditioning of rat heart with monophosphoryl lipid A: a role for nitric oxide. J Pharmacol Exp Ther 285: 1274–1279, 1998.[Abstract/Free Full Text]
  41. Wang Y, Guo Y, Zhang SX, Wu WJ, Wang J, Bao W, and Bolli R. Ischemic preconditioning upregulates inducible nitric oxide synthase in cardiac myocytes. J Mol Cell Cardiol 34: 5–15, 2002.[CrossRef][Web of Science][Medline]
  42. Xuan YT, Tang XL, Qiu Y, Banerjee S, Takano H, Han H, and Bolli R. Biphasic response of cardiac NO synthase isoforms to ischemic preconditioning in conscious rabbits. Am J Physiol Heart Circ Physiol 279: H2360–H2371, 2000.[Abstract/Free Full Text]
  43. Yang XM, Krieg T, Cui L, Downey JM, and Cohen MV. NECA and bradykinin at reperfusion reduce infarction in rabbit hearts by signaling through PI3K, ERK, and NO. J Mol Cell Cardiol 36: 411–421, 2004.[CrossRef][Web of Science][Medline]
  44. Yoshida T, Engelman RM, Engelman DT, Rousou JA, Maulik N, Sato M, Elliott GT, and Das DK. Preconditioning of swine heart with monophosphoryl lipid A improves myocardial preservation. Ann Thorac Surg 70: 895–900, 2000.[Abstract/Free Full Text]
  45. Zhao L, Weber PA, Smith JR, Comerford ML, and Elliott GT. Role of inducible nitric oxide synthase in pharmacological "preconditioning" with monophosphoryl lipid A. J Mol Cell Cardiol 29: 1567–1576, 1997.[CrossRef][Web of Science][Medline]
  46. Zhao TC, Hines DS, and Kukreja RC. Adenosine-induced late preconditioning in mouse hearts: role of p38 MAP kinase and mitochondrial KATP channels. Am J Physiol Heart Circ Physiol 280: H1278–H1285, 2001.[Abstract/Free Full Text]
  47. Zhao T, Xi L, Chelliah J, Levasseur JE, and Kukreja RC. Inducible nitric oxide synthase mediates delayed myocardial protection induced by activation of adenosine A1 receptors: evidence from gene-knockout mice. Circulation 102: 902–907, 2000.[Abstract/Free Full Text]



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