AJP - Heart Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 280: H1278-H1285, 2001;
0363-6135/01 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (34)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhao, T. C.
Right arrow Articles by Kukreja, R. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhao, T. C.
Right arrow Articles by Kukreja, R. C.
Vol. 280, Issue 3, H1278-H1285, March 2001

Adenosine-induced late preconditioning in mouse hearts: role of p38 MAP kinase and mitochondrial KATP channels

Ting C. Zhao, Denise S. Hines, and Rakesh C. Kukreja

Division of Cardiology, Department of Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We investigated the role of p38 mitogen-activated protein kinase (MAPK) phosphorylation and opening of the mitochondrial ATP-sensitive K+ [(KATP)mito] channel in the adenosine A1 receptor (A1AR)-induced delayed cardioprotective effect in the mouse heart. Adult male mice were treated with vehicle (5% DMSO) or the A1AR agonist 2-chloro-N6-cyclopentyladenosine (CCPA; 0.1 mg/kg ip). Twenty-four hours later, hearts were subjected to 30 min of global ischemia and 30 min of reperfusion in the Langendorff mode. Genistein or SB-203580 (1 mg/kg ip) given 30 min before CCPA treatment was used to block receptor tyrosine kinase or p38 MAPK phosphorylation, respectively. 5-Hydroxydecanoate (5-HD; 200 µM) was used to block (KATP)mito channels. CCPA produced marked improvement in left ventricular function, which was partially blocked by SB-203580 and 5-HD and completely abolished with genistein. CCPA caused a reduction in infarct size (12.0 ± 2.0 vs. 30.3 ± 3.0% in vehicle), which was blocked by genistein (29.4 ± 2.3%), SB-203580 (28.3 ± 2.6%), and 5-HD (33.9 ± 2.4%). CCPA treatment also caused increased phosphorylation of p38 MAPK during ischemia, which was blocked by genistein, SB-203580, and 5-HD. The results suggest that A1AR-triggered delayed cardioprotection is mediated by p38 MAPK phosphorylation. Blockade of cardioprotection with 5-HD concomitant with decrease in p38 MAPK phosphorylation suggests a potential role of (KATP)mito channel opening in phosphorylation and ensuing the late preconditioning effect of A1AR.

ischemia; reperfusion myocardial infarction; adenosine; ATP-sensivitive potassium channel; signaling transduction; mitogen-activated protein


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

BRIEF ISCHEMIA before a second sustained ischemia decreases the size of myocardial infarction (26). This cardioprotective phenomenon, known as ischemic preconditioning, has been classified into two temporally distinct phases: an early phase, which wanes within 2-4 h, and a late phase, which manifests 24-72 h later (2). Intense efforts are being made to discover the novel triggers and effectors of preconditioning that could potentially be developed to mimic the effect of ischemic preconditioning. Adenosine is one such agent that has been proposed to be an important trigger of the early and delayed preconditioning (4, 34). However, the underlying intracellular signaling mechanisms of protection induced by this agent are not fully understood. We (37) recently demonstrated that the adenosine A1 receptor (A1AR) agonist 2-chloro-N6-cyclopentyladenosine (CCPA)-induced late preconditioning was due to enhanced synthesis of nitric oxide (NO) in the CCPA-treated heart. This late ischemic protection was abolished by S-methylthiourea, a selective pharmacological inhibitor of inducible NO synthase (iNOS), and was absent in the mouse with targeted ablation of the iNOS gene (37). Because NO has a role in the opening of the mitochondrial ATP-sensitive K+ [(KATP)mito] channel (32), recently, we and others (3-6) demonstrated that the late protection due to A1AR stimulation or ischemic preconditioning was abolished by glibenclamide and 5-hydroxydecanoate (5-HD), the blockers of the KATP channel. However, the cellular signaling pathways potentially modulating the KATP channel and late preconditioning after A1AR activation need further investigation.

The protein tyrosine kinase-mitogen-activated protein kinase (MAPK)-regulated pathway plays an important role for the control of cell growth and differentiation (8). More recently, evidence has been obtained that supports a role for the involvement of tyrosine kinase in early classic preconditioning (22, 35) and late preconditioning (15, 28). Since a novel member of the MAPK family, p38 MAPK, was cloned (11), at least six isoforms of p38 MAP kinase have been described (19). p38 MAPK is activated by dual phosphorylation on a Thr-Gly-Tyr motif in response to endotoxin, cytokines, physical stress (such as hyperosmolarity), and chemical stress (such as hydrogen peroxide) (11). Most interestingly, p38 MAPK has been shown to be activated on exposure to adenosine in perfused rat hearts, which is consistent with MAPK involvement in adenosine-mediated ischemic preconditioning (12). Tyrosine kinase-induced phosphorylation of p38 MAP kinase was shown by preconditioning (22), and the level of phosphorylation of the activation site of p38 MAPK is specifically increased during ischemia (35). A recent study (10) suggested that transient activation of A1AR caused a significant rise in the activity of p38 MAPK 24 h later in the rabbit heart. In addition, the delayed cardioprotection due to A1AR stimulation is abrogated by either the protein kinase C inhibitor chelerythrine or the receptor tyrosine kinase inhibitor lavendustin A.

Considering the fact that late preconditioning induced by A1AR is mediated by opening of KATP channels and is also associated with p38 MAPK phosphorylation, the relationship of these two processes is currently unknown. Although the association of phosphorylation of p38 MAP kinase with A1AR-induced late preconditioning has been reported, it is not known whether such a protective effect is abrogated by the selective MAPK inhibitor SB-203580 in the intact heart (10). SB-203580 is a pyridinylimidazole compound that inhibits the catalytic activity of p38 MAPK by competitive binding in the ATP pocket and has been found to be equipotent against p38-alpha and p38-beta (21). In the present study, we sought to investigate the direct cause and effect relationship of p38 MAPK phosphorylation with A1AR-induced late preconditioning in the mouse heart. Our second goal was to show whether the abrogation of A1AR-induced late cardioprotection with the (KATP)mito channel inhibitor 5-HD is also associated with inhibition of p38 MAPK phosphorylation during ischemia. These investigations were carried out in our well-established model of the isolated perufused mouse heart as reported previously (36, 38).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Adult outbred male mice (IRC Strain) of 30-40 g body wt were supplied by Harlan (Indianapolis, IN). All animal experiments were conducted under the Guidelines on Human Use and Care of Laboratory Animals for Biomedical Research published by the NIH and according to the experimental protocols approved by the Animal Welfare Committee of the Medical College of Virginia Commonwealth University.

Chemicals. CCPA, 5-HD, and SB-203580 were obtained from Research Biochemicals International (Natick, MA); gel electrophoresis supplies were obtained from Bio-Rad Laboratories (Hercules, CA); and genistein and all other chemicals were obtained from Sigma (St. Louis, MO).

Heart perfusion. All animals were anesthetized with a lethal intraperitoneal injection of pentobarbital sodium (100 mg/kg with 33 units heparin). Hearts were rapidly excised and arrested in ice-cold Krebs-Henseleit buffer. They were then cannulated via the ascending aorta for retrograde perfusion by the Langendorff method using Kreb-Henseleit buffer containing (in mM) 110 NaCl, 4.7 KCl, 1.2 MgSO4 · 7H2O, 2.5 CaCl2 · 2H2O, 11 glucose, 1.2 KH2PO4, 25 NaHCO3, and 0.5 EDTA. The buffer, aerated with 95% O2-5% CO2 to give a pH of 7.4 at 37°C, was perfused at a constant pressure of 55 mmHg. A small incision was made at the base of the pulmonary artery to drain coronary effluent.

Left ventricular function. A left atrial incision was made to expose the mitral annulas, through which a tiny balloon was passed into the left ventricle (LV). A water-filled latex balloon, attached to the tip of polyethylene tubing, was then sufficiently inflated to provide a LV end-diastolic pressure (LVEDP) of <10 mmHg, which was measured by means of a disposable Gould pressure transducer. LV pressure and heart rate were recorded using a RF11 A recorder (Sensor Medics, Anaheim, CA). LV maximum changes in positive and negative pressure over time (+dP/dtmax and -dP/dtmax, respectively) were recorded using a Heart Performance Analyzer (Micro-Med; Louisville, KY). Cardiac contractile function, the rate-pressure product (RPP), was expressed as being the product of LV developed pressure (LVDP) and heart rate; LVDP was calculated by subtracting LVEDP from LV systolic pressure.

Experimental protocols. Mice were randomized to receive different treatments by intraperitoneal injection of an agonist, antagonist, or vehicle 24 h before euthanization. The mice were then assigned to one of the following nine groups. 1) CCPA (n = 7): pretreatment with CCPA (0.1 mg/kg ip). 2) Vehicle (n = 9): pretreatment with 5% DMSO (0.1 ml/kg ip). 3) Vehicle + CCPA (n = 9): same as CCPA group except that DMSO was injected 30 min before CCPA. 4) Genistein (n = 7): pretreatment with genistein alone (1 mg/kg ip). 5) Genistein + CCPA (n = 9): same as CCPA group except that genistein (1 mg/kg ip) was injected 30 min before CCPA. 6) SB-203580 (n = 6): pretreatment with SB-203580 (1 mg/kg ip). 7) SB-203580 + CCPA (n = 9): same as the SB-203580 group except that SB-203580 was injected 30 before CCPA. 8) CCPA + 5-HD (n = 6): same as CCPA group except that 5-HD (200 µM) was given in 0.9% saline through the drug line 5 min before ischemia. 9) Vehicle + 5-HD (n = 5): same as CCPA + 5-HD group except that the animals were treated with the vehicle in lieu of CCPA.

After 30 min of equilibration, the hearts were subjected to 30 min of global ischemia and 30 min of reperfusion. The protocol is shown in Fig. 1.


View larger version (24K):
[in this window]
[in a new window]
  		Fig. 1.   Experimental groups and protocol. See METHODS for groups and treatment. CCPA, 2-chloro-N6-cyclopentyladenosine; 5-HD, 5-hydroxydecanoate.

Measurements of myocardial infarct size. At the end of the ischemia-reperfusion, the hearts were removed from the Langendorff perfusion apparatus and immediately weighed, frozen, and stored in a freezer for 24 h. The frozen hearts were then cut from apex to base into six to seven transverse slices of ~0.8 mm in thickness and incubated in 10% triphenyltetrazolium chloride for 30 min. After staining, the slices were fixed in 10% formaldehyde for 40 min before measurement of the infarct area by computer morphometry (Bioquant system, R&M Biometrics). The infarct size was calculated and presented as the percentage of risk area, defined as the sum of total ventricular area minus cavities.

A subset of animals was treated with various drugs solely for the purpose of measuring the p38 MAPK phosphorylation during ischemic period. The mice were assigned into one of the following groups. 1) Vehicle (DMSO): mice pretreated with 5% DMSO (ip) 24 h before ischemia. 2) CCPA: mice pretreated with CCPA 24 h before ischemia (0.1 mg/kg ip). 3) Genistein + CCPA: mice pretreated with genistein (1 mg/kg ip) 30 min before CCPA treatment, as in the CCPA group. 4) SB-203580 + CCPA: mice pretreated with SB-203580 (1 mg/kg ip) 30 min before CCPA. 5) CCPA + 5-HD: 5-HD (200 µM) given into perfusate 5 min before sustained ischemia in CCPA-treated mice.

The hearts were then subjected to global ischemia for 5, 10, 15 , 20, or 30 min, respectively.

Tissue preparation. After the indicated period of global ischemia, as described in the above groups, hearts were immediately frozen in liquid N2. The tissue was ground and suspended in 1 ml of lysis buffer containing 50 mM Tris · HCl (pH 7.4), 0.1 mM sodium orthovanadate, 50 mM sodium fluoride, 150 mM sucrose, 1 mM phenylmethylsulfonyl fluoride, 5 mM EDTA, 5 mM EGTA, 2 µg/ml leupeptin, 2 µg/ml aprotinin, and 5 µg/ml pepstatin A. Mixtures were homogenized with a Brinkman polytron and microcentrifuged at 14,000 rpm for 10 min. The protein content of the supernatant was determined using the detergent compatible-protein assay (Bio-Rad).

Western blot. Western blot analysis was performed as described previously with some modification (13). The proteins (40 µg), separated by SDS-PAGE (10% polyacrylamide), were transferred to a nitrocellulose membrane (0.2 µM) by tank transfer (Bio-Rad) for 2 h at 200 mA. The membranes were first probed with phospho-specific p38 MAPK (Thr 180/Tyr 182) antibody (New England Biolabs; Beverly, MA), after which they were stripped in 100 mM 2-mercaptoetanol, 2% SDS, and 62.5 mM Tris · HCl (pH 6.7) for 30 min at 56°C, blocked, and reprobed with nonspecific p38 MAPK antibody (for total p38 MAPK; Santa Cruz Biotechnology; Santa Cruz, CA). Briefly, membranes were either blocked with 5% BSA and 0.1% Tween 20 in Tris-buffered saline for 2 h by overnight incubation at 4°C with phospho-specific p38 MAPK antibody (1:1,000 dilution) or blocked with 5% nonfat dry milk in Tris-buffered saline overnight at 4°C. The membranes were then incubated for 1 h with p38 MAPK antibody (1:1,000 dilution). The membranes were washed with Tris-buffered saline containing 0.1% Tween 20 and incubated with an anti-rabbit horseradish peroxidase-linked antibody (Amersham Pharmacia Biotech; Piscataway, NJ) diluted 1:500 for 1 h at room temperature followed by detection with chemiluminescence (Amersham ECL Western blotting detection reagent).

Statistical analysis. The results are expressed as means ± SE. Differences among the groups were analyzed by one-way analysis of variance (ANOVA). Statistical differences were considered significant if the P value was <0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Infarct size. Myocardial infarct size was 30.3 ± 3.0% of the risk zone in the vehicle-treated hearts. Treatment with CCPA reduced infarct size to 12.2 ± 2.5% (P < 0.05; Fig. 2). The infarct size in the vehicle + CCPA group was 17.0 ± 2.9%, which was not different compared with the CCPA group. Genistein and SB-203580 completely abolished the protective effect of CCPA, as indicated by increase in the infarct size to 29.4 ± 2.3 and 28.3 ± 2.6%, respectively (P < 0.05 vs. 12.2 ± 2.5% in control). The infarct sizes in the genistein- and SB-203580-treated mice were 30.0 ± 1.6 and 28.3 ± 2.6%, respectively, which were indistinguishable from the vehicle group. Infusion of 5-HD before ischemia-reperfusion in CCPA-treated mice resulted in significant increases in the infarct size to 33.9 ± 2.4. Vehicle + 5-HD did not cause a significant change in the infarct size compared with that of the vehicle group. The area at risk for the globally ischemic hearts was not different between groups (data not shown).


View larger version (33K):
[in this window]
[in a new window]
  		Fig. 2.   Bar diagram showing infarct size expressed as percentage of area at risk. Values are means ± SE. *P < 0.05 vs. CCPA and vehicle + CCPA.

LV function. Myocardial functional parameters, such as LV systolic pressure, LVEDP, dP/dtmax, heart rate, and coronary flow during preischemia, were not significantly different among the groups (Table 1). Also, average body and heart weights were similar among all the groups (data not shown). In the ischemic-reperfused heart, LVEDP was significantly lower in the CCPA-treated group as compared with the vehicle-treated control (P < 0.05; Fig. 3A). The CCPA-induced improvement in LVEDP was abrogated by genistein (P < 0.05 vs. CCPA). Genistein itself had no significant effect on LVEDP in the control group (non-CCPA-treated mice). SB-203580 did not abolish the improvement in LVEDP observed in the vehicle-treated CCPA group. In fact, the LVEDP was found to be significantly lower in the SB-203580-treated groups (with or without treatment with CCPA). Similarly, 5-HD failed to block the improvement in postischemic LVEDP when compared with the vehicle-CCPA group (P > 0.05). Also, 5-HD did not have significant effect on the postischemic levels of LVEDP when compared with the vehicle or CCPA-treated mice.

                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Baseline functional parameters



View larger version (50K):
[in this window]
[in a new window]
  		Fig. 3.   Postischemic recovery of left ventricular (LV) function. A: LV end-diastolic pressure (LVEDP, in mmHg); B: rate-pressure product (RPP). Results represent means ± SE. *P < 0.05 vs. CCPA.

Recovery of RPP was 63.9 ± 9.3% in the vehicle control group, increased to 96 ± 7% with CCPA (P > 0.05; Fig. 3B), and was abolished by genistein (52.1 ± 9.5%), SB-203580 (70.2 ± 7.5%), and 5-HD (67.5% ± 2.9), although these differences were nonsignificant compared with CCPA. A similar trend in the changes in LVDP was observed (data not shown). No significant changes in +dP/dtmax or -dP/dtmax was observed among the groups (data not shown).

Also, the heart rate and coronary flow were also not different between the groups (Fig. 4, A and B).


View larger version (64K):
[in this window]
[in a new window]
  		Fig. 4.   Postischemic changes in heart rate (in beats/min; A) and coronary flow (in ml/min; B). Results represent means ± SE.

Western blot analysis of p38 MAPK phosphorylation. The total p38 MAPK protein expression was not different between groups, i.e., the levels detected remained fairly constant throughout the ischemia (Fig. 5, right). However, the phospho-specific p38 MAPK protein increased in hearts pretreated with CCPA during 10, 15, and 20 min of ischemia (Fig. 5, left). The increase in CCPA-induced phospho-p38 MAPK protein was inhibited in mice that were pretreated with genistein as well as SB-203580. Interestingly, similar abolition of phospho-p38 MAPK expression was observed in the CCPA-pretreated heart with infusion of 5-HD 5 min before ischemia. DMSO had no effect on the phosphorylation state of p38 MAPK protein expression.


View larger version (54K):
[in this window]
[in a new window]
  		Fig. 5.   Western blot showing expression of p38 mitogen-activated protien (MAP) kinase. A subset of hearts were treated with vehicle and CCPA ± genistein/SB-203580/5-HD, as described in detail under METHODS. Twenty-four hours later, the hearts were isolated and subjected to ischemia for 5, 10, 15, 20, and 30 min of global ischemia. The Western blots of each group were incubated with an antibody against phospho-p38 MAP kinase (left). The blots were stripped and probed again with an antibody against p38 MAP kinase (right).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Salient findings. The main goal of this investigation was to demonstrate the cause and effect relationship of p38 MAPK phosphorylation in A1AR receptor-induced delayed cardioprotection in the mouse heart. In addition, we wanted to know whether the opening of the (KATP)mito channel is correlated with p38 MAPK phosphorylation. Our results show that selective activation of A1AR with CCPA reduced postischemic infarct size, improved recovery of LVEDP and RPP, and caused p38 MAPK phosphorylation in the ischemic heart 24 h after the receptor stimulation. The receptor tyrosine kinase inhibitor genistein as well as the selective p38 MAPK inhibitor SB-203580, when given before CCPA treatment, abolished the delayed cardioprotective effect as well as inhibited p38 MAPK phosphorylation during ischemia. Furthermore, selective blockade of the (KATP)mito channel with 5-HD not only abrogated the A1AR-induced late cardioprotection but also inhibited p38 MAPK phosphorylation. Pharmacological inhibitors (genistein, SB-203580, or 5-HD) were without significant effect on infarct size in mice that were not treated with CCPA. Taken together, our data suggest that p38 MAPK phosphorylation and opening of the (KATP)mito channel play essential role in A1AR-induced delayed cardioprotective effect in the isolated perfused mouse heart.

Tyrosine kinase signaling in preconditioning. The tyrosine kinases are important for the control of cell growth and differentiation and are rapidly phosphorylated on tyrosine residues in response to various stimuli, leading to the activation of the MAPK signaling pathway (8). In the isolated perfused rat heart, p38 MAPK has been shown to be activated during global ischemia and sustained throughout reperfusion (9, 20). Involvement of p38 MAPK signaling pathway in the early phase of preconditioning has been extensively investigated, although the results are controversial. Maulik et al. (22) first suggested the involvement of tyrosine kinase-phospholipase D as a potential signaling pathway for ischemic preconditioning. Weinbrenner et al. (35) also showed decreased phosphorylation of p38 MAP kinase during ischemia in nonpreconditioned hearts, but phosphorylation was enhanced several-fold after 10 and 20 min of ischemia in the preconditioned hearts. Furthermore, when protection from ischemic preconditioning was blocked by adenosine receptor antagonist, the increased phosphorylation of p38 MAPK during ischemia also disappeared. In addition, anisomycin, an activator of the p38 MAPK pathway, was found to be as protective as preconditioning (35). Sakamoto et al. (31) also demonstrated partial blockade of the cardioprotective effects of CCPA with SB-203580 in the isolated perfused rat heart. On the other hand, Barancik et al. (1) suggested a negative role of the p38 MAPK pathway during ischemic activation of p38 MAPKs. Ma et al. (20) showed that administration of SB-203580 decreased myocardial apoptosis and improved postischemic cardiac function in the isolated perfused heart. In the present study, we observed significant blockade of A1AR-induced late preconditioning with SB-203580 as well as genistein, which also correlated with decreased expression of phosphorylated p38 MAPK during ischemia in the mouse heart. Although Dana et al. (10) also demonstrated the role of p38 MAPK in A1AR-induced protection, these studies arrived at this conclusion by showing association of p38 MAPK phosphorylation 24 h later and its blockade by lavendustin, a relatively selective inhibitor of receptor tyrosine kinase. These studies were carried out in a rabbit model of myocardial infarction, and LV function was not measured. However, the present study demonstrates the protective effect p38 MAPK phosphorylation in a mouse model with multiple end points, i.e., infarct size as well as LV function. This model could be useful in further evaluation of genes involved in A1AR-induced late preconditioning.

In the present study, we observed an increase in phosphorylation of p38 MAPK only during ischemia in the hearts treated with CCPA, whereas this was absent in the nontreated hearts. Maulik et al. (23) showed that pretreatment with genistein attenuated the increase in both MAPK and MAPK-activated protein kinase (MAPKAPK)-2 activities, which occurred in the preconditioned hearts. Another study (20) showed increased p38 MAPK activity in nonpreconditioned hearts subjected to ischemia-reperfusion, whereas Weinbrenner et al. (35) observed p38 MAPK phosphorylation only in preconditioned hearts.

From the present study, it appears that p38 MAPK is the mediator of the delayed cardioprotective effect of CCPA. It was recently proposed that MAPKs exhibited a biphasic activation on exposure to certain stimuli (21). Haq et al. (12) showed that infusion of adenosine resulted in rapid activation of p38 MAPK that was maximal at 5 min and declined thereafter. We did not measure the initial time course of p38 phosphorylation after CCPA treatment in the present study. However, from the results presented in this paper and those of others (10), it appears that p38 MAPK phosphorylation after 24 h of CCPA treatment is important in mediating the late phase of preconditioning.

Role of (KATP)mito channel. In the present study, we also observed blockade of the delayed preconditioning effect of CCPA with 5-HD. Several previous studies from this laboratory and others (5, 6, 13, 25, 29) have shown that late preconditioning induced by sublethal ischemia, heat shock, and pharmacological agents (such as CCPA and monophosphoryl lipid A) are mediated by opening of the KATP channel in the rabbit heart. It has been suggested that (KATP)mito channels are the effectors of preconditioning, because diazoxide, a selective opener of this channel, induced an early as well as a delayed cardioprotective effect (27, 33), which was abolished by 5-HD. An interesting aspect of this study is that 5-HD, when given before ischemia, not only abolished the late cardioprotective effect of CCPA but also inhibited p38 MAPK phosphorylation during ischemia in this group. These data suggest that p38 MAPK phosphorylation is an essential step in mediating the late cardioprotective effect due to opening of the (KATP)mito channel. The precise interrelationship of p38 MAPK phosphorylation with the opening of the channel is not clear from the present study. A recent study (28a) demonstrated that pharmacological preconditioning induced by selective opening of the (KATP)mito channel with diazoxide is blocked by the free radical scavenger mercaptopropionyl glycine. It is possible that activation of cardiac (KATP)mito channels during ischemia in the CCPA-treated hearts may have a role in the generation of reactive oxygen species, which could potentially phosphorylate p38 MAPK. Further investigations are needed to understand whether CCPA-induced activation of the (KATP)mito channel during the late phase is blocked by antioxidants.

Role of NO and heat shock protein27. There are other possible consequences of MAPK phosphorylation also. For example, the 27-kDa heat shock protein (HSP27) is phosphorylated by MAPKAPK-2 and -3, which are the kinases downstream of p38 MAPK (16, 24, 30). HSP27 is known to exert its cardioprotective effect by stabilizing the microfilaments (18). Dana et al. (10) suggested an important role for both protein kinase C and receptor tyrosine kinase as mediators of late preconditioning against infarction after A1AR activation. It was proposed that the p38/HSP27 pathway was a potential distal effector of protection. In the present study, we did not measure the phosphorylated state of HSP27. However, it is quite possible that the inhibition of p38 MAPK phosphorylation with SB-203580 may have prevented the phosphorylation of HSP27 during ischemia, thereby promoting disruption of more actin filaments, resulting in enhanced fragility. Conversely, it has been shown that anisomycin, which activates p38 MAPK with subsequent phosphorylation of HSP27, could have prevented actin filament disruption (14). Additionally, p38 MAPK phosphorylation may have activated iNOS, resulting in enhanced NO release. We (37) recently reported that A1AR activation-induced delayed protection was mediated by an iNOS-sensitive mechanism. p38 MAPK is known to regulate iNOS gene expression in endotoxin-stimulated primary glial cultures (7). The inhibition of p38 MAPK partially reduced interleukin stimulation of iNOS protein and iNOS mRNA in cardiac myocytes, suggesting that interleukin induction of iNOS synthesis depends on p38 MAPK signaling pathways (17).

In conclusion, we have shown that selective activation of A1AR with CCPA induced late cardioprotective effect 24 h after the receptor stimulation in the mouse heart. This protective effect was blocked by the receptor tyrosine kinase inhibitor genistein and the p38 MAPK inhibitor SB-203580. In addition, A1AR receptor stimulation caused increased phosphorylation of p38 MAPK during ischemia 24 h later, which was also blocked by genistein as well as SB-203580. These data suggest a direct cause and effect relationship of receptor tyrosine kinase or, more specifically, p38MAPK phosphorylation in mediating A1AR-induced delayed cardioprotection. Furthermore, the blockade of A1AR-induced delayed cardioprotection with 5-HD also suggests an obligatory role of (KATP)mito channels. It is possible that reduced oxygen species generated as a result of (KATP)mito channel opening may have a role in p38 MAPK phosphorylation. The net protective effect may be due to combination of factors including generation of NO/reduced oxygen species, opening of the (KATP)mito channel, and phosphorylation of HSP27. Further studies are necessary to elucidate the precise relationship of these mediators and effectors in late preconditioning induced by "pharmacological" activation of A1AR.


    ACKNOWLEDGEMENTS

This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-51045 and HL-59469 (to R. C. Kukreja). D. Hines was supported by a fellowship from the National Heart, Lung, and Blood Institute Training Grant HL-07537.


    FOOTNOTES

Address for reprint requests and other correspondence: R. C. Kukreja, Div. of Cardiology, Box 281, Medical College of Virginia, Virginia Commonwealth Univ., Richmond, VA 23298 (E-mail: rakesh{at}hsc.vcu.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.

Received 31 July 2000; accepted in final form 28 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Barancik, M, Hatun P, Strohm C, Kilian S, and Schaper W. Inhibition of the cardiac p38-MAPK pathway by SB-203580 delays ischemic cell death. J Cardiovasc Pharmacol 35: 474-483, 2000[ISI][Medline].

2.   Baxter, GF, Goma FM, and Yellon DM. Characterisation of the infarct-limiting effect of delayed preconditioning: time course and dose-dependency studies in rabbit myocardium. Basic Res Cardiol 92: 159-167, 1997[ISI][Medline].

3.   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].

4.   Baxter, GF, and Yellon DM. ATP-sensitive K+ channels mediate the delayed cardioprotective effect of adenosine A1 receptor activation. J Mol Cell Cardiol 31: 981-989, 1999[ISI][Medline].

5.   Bernardo, NL, D'Angelo M, Okubo S, Joy A, and Kukreja RC. Second window of ischemic preconditioning is mediated by opening of ATP-sensitive potassium channels in the rabbit heart. Am J Physiol Heart Circ Physiol 276: H1323-H1330, 1999[Abstract/Free Full Text].

6.   Bernardo, NL, Okubo S, Maaieh M, Wood MA, and Kukreja RC. Delayed preconditioning with adenosine is mediated by opening of ATP-sensitive K+ chennels in rabbit heart. Am J Physiol Heart Circ Physiol 277: H128-H135, 1999[Abstract/Free Full Text].

7.   Bhat, NR, Zhang P, Lee JC, and Hogan EL. Extracellular signal-regulated kinase and p38 subgroups of mitogen-activated protein kinases regulate inducible nitric oxide synthase and tumor necrosis factor-alpha gene expression in endotoxin-stimulated primary glial cultures. J Neurosci 18: 1633-1641, 1998[Abstract/Free Full Text].

8.   Blumer, KJ, and Johnson GL. Diversity in function and regulation of MAP kinase pathways. Trends Biochem Sci 19: 236-240, 1994[ISI][Medline].

9.   Bogoyevitch, MA, Gillespie-Brown J, Ketterman AJ, Fuller SJ, Ben-Levy R, Ashworth A, Marshall CJ, and Sugden PH. Stimulation of the stress-activated mitogen-activated protein kinase subfamilies in perfused heart: p38/RK MAPK and c-Jun N-terminal kinases are activated by ischemia/reperfusion. Circ Res 79: 162-173, 1996[Abstract/Free Full Text].

10.   Dana, A, Skarli M, Papakrivopoulou J, and Yellon DM. Adenosine A(1) 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.   Han, J, Lee JD, Bibbs L, and Ulevitch RJ. A new MAP kinase targeted by endotoxin and hyperosmolarity in mamalian cells. Science 267: 389-398, 1994.

12.   Haq, SE, 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[ISI][Medline].

13.   Hoag, JB, Qian Y-Z, Nayeem MA, D'Angelo M, and Kukreja RC. ATP-sensitive potassium channel mediates delayed ischemic protection by heat stress in rabbit heart. Am J Physiol Heart Circ Physiol 273: H861-H868, 1997[Abstract/Free Full Text].

14.   Huot, J, Houle F, Spitz DR, and Landry J. HSP27 phosphorylation-mediated resistance against actin fragmentation and cell death induced by oxidative stress. Cancer Research 56: 273-279, 1996[Abstract/Free Full Text].

15.   Imagawa, J-I, Baxter GF, and Yellon DM. Genistein, a tyrosine kinase inhibitor, blocks the "second window of protection" 48 h after ischemic preconditioning in the rabbit. J Mol Cell Cardiol 29: 1885-1893, 1997[ISI][Medline].

16.   Knauf, U, Jacob U, Engel K, Buchner J, and Gaestel M. Stress- and mitogen-induced phosphorylation of the small heat shock protein Hsp25 by MAPKAP kinase 2 is not essential for chaperone properties and cellular thermoresistance. EMBO J 13: 54-60, 1994[ISI][Medline].

17.   Lapointe, MC, and Isenovic E. Interleukin-1beta regulation of inducible nitric oxide synthase and cyclooxygenase-2 involves the p42/44 and p38 MAPK signaling pathways in cardiac myocytes. Hypertension 33: 276-282, 1999[Abstract/Free Full Text].

18.   Lavoie, JN, Gingras-Breton G, Tanguay RM, and Landry J. Induction of Chinese hamster HSP27 gene expression in mouse cells confers resistance to heat shock. HSP27 stabilization of the microfilament organization. J Biol Chem 268: 3420-3429, 1993[Abstract/Free Full Text].

19.   Lazou, A, Sudgen PH, and Clerk A. Activation of mitogen-activated protein kinases (p38-MAPKs, SAPKs/JNKs and ERKs) by the G-protein-coupled receptor agonist phenylephrine in the perfused rat heart. Biochem J 332: 459-465, 1998.

20.   Ma, XL, Kumar S, Gao F, Louden CS, Lopez BL, Christopher TA, Wang C, Lee JC, Feuerstein GZ, and Yue TL. Inhibition of p38 mitogen-activated protein kinase decreases cardiomyocyte apoptosis and improves cardiac function after myocardial ischemia and reperfusion. Circulation 99: 1685-1691, 1999[Abstract/Free Full Text].

21.   Magasaka, Y, and Nakamura K. Modulation of the heat-induced activation of mitogen-activated protein (MAP) kinase by quercetin. Biochem Pharmacol 56: 1151-1155, 1998[ISI][Medline].

22.   Maulik, N, Watanabe M, Zu YL, Huang CK, Cordis GA, Schley JA, and Das DK. Ischemic preconditioning triggers the activation of MAP kinases and MAPKAP kinase 2 in rat hearts. FEBS Lett 396: 233-237, 1996[ISI][Medline].

23.   Maulik, N, Yoshida T, Zu YL, Sato M, Banerjee A, and Das DK. Ischemic preconditioning triggers tyrosine kinase signaling: a potential role for MAPKAP kinase 2. Am J Physiol Heart Circ Physiol 275: H1857-H1864, 1998[Abstract/Free Full Text].

24.   McLaughlin, MM, Kumar S, McDonnell PC, Van Horn S, Lee JC, Livi GP, and Young PR. Identification of mitogen-activated protein (MAP) kinase-activated protein kinase-3, a novel substrate of CSBP p38 MAP kinase. J Biol Chem 271: 8488-8492, 1996[Abstract/Free Full Text].

25.   Mei, DA, Elliott GT, and Gross GJ. KATP channels mediate late preconditioning against infarction produced by monophosphoryl lipid A. Am J Physiol Heart Circ Physiol 271: H2723-H2729, 1996[Abstract/Free Full Text].

26.   Murry, CE, Jennings RB, and Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 74: 1124-1136, 1986[Abstract/Free Full Text].

27.   Ockaili, R, Emani VR, Okubo S, Brown M, Krottapalli K, and Kukreja RC. Opening of mitochondrial KATP channel induces early and delayed cardioprotective effect: role of nitric oxide. Am J Physiol Heart Circ Physiol 277: H2425-H2434, 1999[Abstract/Free Full Text].

28.   Okubo, S, Bernardo NL, Elliott GT, Hess ML, and Kukreja RC. Tyrosine kinase signaling in action potential shortening and expression of HSP72 in late preconditioning. Am J Physiol Heart Circ Physiol 279: H2269-H2276, 2000[Abstract/Free Full Text].

28a.   Pain, T, Yang XM, Cvitz SD, Nakano A, Liu GS, Heusch G, Cohn MV, and Downey JM. Opening of mitochondrial K(ATP) channels triggers the preconditioned state by generating free radicals. Circ Res 87: 460-466, 2000[Abstract/Free Full Text].

29.   Pell, TJ, Yellon DM, Goodwin RW, and Baxter GF. Myocardial ischemic tolerance following heat stress is abolished by ATP-sensitive potassium channel blockade. Cardiovasc Drugs Ther 11: 679-686, 1997[ISI][Medline].

30.   Rouse, J, Cohen P, Trigon S, Morange M, Alonso-Llamazares A, Zamanillo D, Hunt T, and Nebreda AR. A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins. Cell 78: 1027-1037, 1994[ISI][Medline].

31.   Sakamoto, K, Urushidani T, and Nagao T. Translocation of HSP27 to sarcomere induced by ischemic preconditioning in isolated rat hearts. Biochem Biophys Res Commun 269: 137-142, 2000[ISI][Medline].

32.   Sasaki, N, Sato T, Ohler A, O'Rourke B, and Marban E. Activation of mitochondrial ATP-dependent potassium channels by nitric oxide. Circulation 101: 439-445, 2000[Abstract/Free Full Text].

33.   Takashi, E, Wang Y, and Ashraf M. Activation of mitochondrial KATP channel elicits late preconditioning against myocardial infarction via protein kinase C signaling pathway. Circ Res 85: 1146-1153, 1999[Abstract/Free Full Text].

34.   Tsuchida, A, Liu GS, Wilborn WH, and Downey JM. Pretreatment with the adenosine A1 selective agonist, 2-cyclopentyladenosine (CCPA), causes a sustained limitation of infarct size in rabbits. Cardiovasc Res 27: 652-656, 1993[ISI][Medline].

35.   Weinbrenner, C, Liu GS, Cohen MV, and Downey JM. Phosphorylation of tyrosine 182 of p38 mitogen-activated protein kinase correlates with the protection of preconditioning in the rabbit heart. J Mol Cell Cardiol 29: 2383-2391, 1997[ISI][Medline].

36.   Xi, L, Jarrett NC, Hess ML, and Kukreja RC. Myocardial ischemia/reperfusion injury in the inducible nitric oxide synthase knockout mice. Life Sci 65: 935-945, 1999[ISI][Medline].

37.   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].

Am J Physiol Heart Circ Physiol 280(3):H1278-H1285
0363-6135/01 $5.00 Copyright © 2001 the American Physiological Society


This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
J. Zheng, R. Wang, E. Zambraski, D. Wu, K. A. Jacobson, and B. T. Liang
Protective roles of adenosine A1, A2A, and A3 receptors in skeletal muscle ischemia and reperfusion injury
Am J Physiol Heart Circ Physiol, December 1, 2007; 293(6): H3685 - H3691.
[Abstract] [Full Text] [PDF]

Home page
 ICVTSHome page
E. Hochhauser, D. Leshem, O. Kaminski, Y. Cheporko, B. A. Vidne, and A. Shainberg
The protective effect of prior ischemia reperfusion adenosine A1 or A3 receptor activation in the normal and hypertrophied heart
Interactive CardioVascular and Thoracic Surgery, June 1, 2007; 6(3): 363 - 368.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
Y. Guo, A. B. Stein, W.-J. Wu, X. Zhu, W. Tan, Q. Li, and R. Bolli
Late preconditioning induced by NO donors, adenosine A1 receptor agonists, and {delta}1-opioid receptor agonists is mediated by iNOS
Am J Physiol Heart Circ Physiol, November 1, 2005; 289(5): H2251 - H2257.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
R. D. Lasley, B. J. Keith, G. Kristo, Y. Yoshimura, and R. M. Mentzer Jr.
Delayed adenosine A1 receptor preconditioning in rat myocardium is MAPK dependent but iNOS independent
Am J Physiol Heart Circ Physiol, August 1, 2005; 289(2): H785 - H791.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
L. Willems, K. J. Ashton, and J. P. Headrick
Adenosine-mediated cardioprotection in the aging myocardium
Cardiovasc Res, May 1, 2005; 66(2): 245 - 255.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
C. Ballard-Croft, G. Kristo, Y. Yoshimura, E. Reid, B. J. Keith, R. M. Mentzer Jr., and R. D. Lasley
Acute adenosine preconditioning is mediated by p38 MAPK activation in discrete subcellular compartments
Am J Physiol Heart Circ Physiol, March 1, 2005; 288(3): H1359 - H1366.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
A. Das, R. Ockaili, F. Salloum, and R. C. Kukreja
Protein kinase C plays an essential role in sildenafil-induced cardioprotection in rabbits
Am J Physiol Heart Circ Physiol, April 1, 2004; 286(4): H1455 - H1460.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
J. P. Headrick, B. Hack, and K. J. Ashton
Acute adenosinergic cardioprotection in ischemic-reperfused hearts
Am J Physiol Heart Circ Physiol, November 1, 2003; 285(5): H1797 - H1818.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
T. C. Zhao and R. C. Kukreja
Protein kinase C-{delta} mediates adenosine A3 receptor-induced delayed cardioprotection in mouse
Am J Physiol Heart Circ Physiol, June 5, 2003; 285(1): H434 - H441.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
H. H. Patel, R. M. Fryer, E. R. Gross, R. A. Bundey, A. K. Hsu, M. Isbell, L. O.V. Eusebi, R. V. Jensen, S. R. Gullans, P. A. Insel, et al.
12-Lipoxygenase in Opioid-Induced Delayed Cardioprotection: Gene Array, Mass Spectrometric, and Pharmacological Analyses
Circ. Res., April 4, 2003; 92(6): 676 - 682.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
J. Peart, L. Willems, and J. P. Headrick
Receptor and non-receptor-dependent mechanisms of cardioprotection with adenosine
Am J Physiol Heart Circ Physiol, February 1, 2003; 284(2): H519 - H527.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
M. Kudo, Y. Wang, M. Xu, A. Ayub, and M. Ashraf
Adenosine A1 receptor mediates late preconditioning via activation of PKC-delta signaling pathway
Am J Physiol Heart Circ Physiol, July 1, 2002; 283(1): H296 - H301.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
J. Han, N. Kim, H. Joo, and E. Kim
Ketamine abolishes ischemic preconditioning through inhibition of KATP channels in rabbit hearts
Am J Physiol Heart Circ Physiol, July 1, 2002; 283(1): H13 - H21.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
Y. Xu, D. Kumar, J. R. B. Dyck, W. R. Ford, A. S. Clanachan, G. D. Lopaschuk, and B. I. Jugdutt
AT1 and AT2 receptor expression and blockade after acute ischemia-reperfusion in isolated working rat hearts
Am J Physiol Heart Circ Physiol, April 1, 2002; 282(4): H1206 - H1215.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (34)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhao, T. C.
Right arrow Articles by Kukreja, R. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhao, T. C.
Right arrow Articles by Kukreja, R. C.

HOME HELP