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Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
Submitted 4 December 2002 ; accepted in final form 3 February 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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1-opioid receptor (DOR) antagonist 7-benzylidenealtrexone.
Simultaneous administration of morphine and CCPA failed to enhance the
infarct-sparing effect of either agonist alone. These data suggest that both
DOR and A1AR-mediated cardioprotection are mitoKATP and
reactive oxygen species dependent. Furthermore, these data suggest that there
are converging pathways and/or receptor cross-talk between A1AR-
and DOR-mediated cardioprotection.
reactive oxygen species; ATP-sensitive potassium channel
-opioid receptor (DOR)
(45,
47,
54). Indeed, blockade of the
DOR abrogates the infarct-sparing effect of ischemic preconditioning
(55). The signal-transduction pathways responsible for both DOR and A1/A3AR-mediated cardioprotection appear to be similar. A1ARs and A3ARs activate phospholipase C and phospholipase D, respectively (44). Activation of the DOR has also been reported to activate phospholipase C (33, 41). Activation of phospholipase C or phospholipase D results in translocation of specific isoforms of protein kinase C (PKC) (59, 61). Translocation of PKC may then lead to opening of a mitochondrial ATP-sensitive K+ (KATP) channel (mitoKATP) after phopshorylation of a mitochondrial site (43). However, this point is controversial because it has also been reported (36, 64) that opening of the mitoKATP channel may be upstream of PKC and that activation of this channel may lead to generation of reactive oxygen species (ROS), which may subsequently activate PKC and various downstream kinases to produce cardioprotection (7).
We and others (7, 47) have previously reported that the infarct-sparing effect of DOR activation is both mitoKATP and ROS dependent; however, there are opposing reports regarding the role of the mitoKATP channel and ROS in adenosine-mediated cardioprotection. Cohen et al. (7) observed that protection afforded by adenosine receptor activation was independent of both mitoKATP channel opening and ROS in isolated rabbit hearts, whereas other investigators reported (19, 63) that mitoKATP may be integral to the cardioprotective effects of adenosine.
Both A1AR and DOR activation appear to mediate protection via
similar pathways. Indeed, there is considerable evidence in the central
nervous system (CNS) that adenosine and opioid receptors are tightly coupled.
Adenosine receptor activation via adenosine kinase inhibition has been shown
to attenuate opiate withdrawal
(26). Furthermore, inhibition
of neuronal Ca2+ channels by opioid- and adenosine-receptor
agonists was mutually exclusive, supporting the possibility of a convergence
for the two stimuli (6).
Adenosine and morphine inhibit Ca2+-dependent neurotransmitter
release, and this inhibition could be blocked by the nonselective adenosine
receptor antagonist theophylline
(20,
53). The adenosine uptake
inhibitor dipyridamole potentiates adenosine- and morphine-mediated inhibition
of neurotransmitter release
(18,
42). Moreover, a recent study
(17) determined that µ- and
-opioid receptor activation increases adenosine concentrations in rat
striatal cell extracts by inhibiting adenosine uptake. These examples suggest
that important links exist between opioids and adenosine, interactions that,
to date, have not been addressed in the heart.
Therefore, the goals of this study were twofold: first, to examine the controversial role of ROS and mitoKATP channels in A1AR- and DOR-mediated cardioprotection and, finally, to determine whether there is a functional coupling of adenosine and opioid receptors in the heart, leading to cardioprotection.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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General surgical preparation. Male Sprague-Dawley rats weighing 250–350 g were used throughout 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. Atelectasis was prevented by the maintenance of a positive end-expiratory pressure of 5–10 mmH2O. Arterial pH, PCO2, and PO2 Were monitored throughout the protocol with the use of a pH/blood gas analyzer (model 995, AVL) 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 with the use of a heating pad.
The right carotid artery was cannulated with polyethylene-50 tubing to measure blood pressure and heart rate via a polyethylene-23 (Gould) pressure transducer connected to a polygraph (model 7, Grass). The right jugular vein was cannulated for delivery of saline or drug infusion. A left thoracotomy was performed at the fifth intercostal space, followed by a pericardiotomy and adjustment of the left atrial appendage to reveal the location of the left coronary artery. A ligature (6-0 prolene) was passed below the left descending vein and coronary artery from the area immediately below the left atrial appendage to the right portion of the left ventricle (LV). The ends of the suture were threaded through a propylene tube to form a snare. Occlusion of the coronary artery and the subsequent regional LV ischemia was introduced by pulling the ends of the suture taut and clamping the snare onto the epicardial surface with a hemostat. Epicardial cyanosis and a subsequent decrease in blood pressure verified coronary artery occlusion. Reperfusion of the heart was initiated via unclamping the hemostat and relieving tension from the snare and was confirmed by visualizing a marked epicardial hyperemic response. Heart rate and blood pressure were allowed to stabilize before the experimental protocols were initiated.
Determination of infarct size. On completion of the experimental 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 heart was excised, and the LV was removed from the remaining tissue and subsequently cut into five thin cross-sectional pieces. This allowed for the delineation of the normal area, which was 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% triphenyltetrazolium chloride stain in 100 mM phosphate buffer (pH 7.4) at 37°C. 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).
Experimental protocols. After equilibration of hemodynamic parameters, the rats were divided into various experimental groups (Fig. 1). Rats were either untreated, treated with agonist alone, antagonist alone, or treated with both agonists and antagonists. Agonists were given as a bolus dose 10 min before the onset of ischemia, whereas antagonists were given 20 min before ischemia, except in the case of 5-hydroxydecanoate (5-HD), which was administered 15 min before agonists due to its short half-life. After treatment, hearts were occluded for 30 min, followed by 90 min of reperfusion before triphenyltetrazolium chloride staining.
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Exclusion criteria. Rats were excluded from data analysis if they exhibited severe hypotension (<30 mmHg systolic 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 protocol groups.
Chemicals. 2-Chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyl-uronamide (Cl-IB-MECA), 2-chloro-N6-cyclopentyladenosine (CCPA), 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), 5-HD, N-(2-mercaptopropionyl)glycine (2-MPG), 3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine carboxylate (MRS-1523), and morphine sulfate were obtained from Sigma (St. Louis, MO). 7-Benzylidenenaltrexone maleate (BNTX) was purchased from Tocris (Ellisville, MO).
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 in any parameter between groups. Significant differences were determined at P < 0.05.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Effect of A1AR and DOR activation and blockade. Because of the hemodynamic effects of CCPA treament (i.e., bradycardia), we attempted to find a dose where cardioprotection was still present, yet the hemodymanic effects were minimal. Three doses (10, 30, and 100 µg/kg) were examined. The effect of CCPA on heart rate was dramatically reduced at 10 µg/kg, whereas the infarct-limiting effect was only slightly reduced from that seen following the two higher doses (Fig. 3). Thus the 10 µg/kg dose of CCPA was chosen for all future studies.
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After 30-min occlusion and 90-min reperfusion, pretreatment with 10 µg/kg CCPA (n = 6) significantly reduced IS (%IS/AAR) compared with untreated rats (P < 0.01 vs. untreated, Fig. 2). To further eliminate the bradycardic effect of CCPA as a possible mechanism leading to the infarct-limiting effect, a subset of rats was electrically paced at 6 Hz. Pacing was achieved by placing two electrodes on the surface of the ventricle. The pacing voltage was kept <5 V, and pacing was maintained throughout the experimental protocol. No differences were noted between unpaced and paced hearts pretreated with 10 µg/kg CCPA (39.9 ± 4.4%, 35.0 ± 1.3% IS/AAR for unpaced and paced hearts, respectively). Morphine (0.3 mg/kg, n = 7), when administered as a bolus dose 10 min before the onset of ischemia, also produced a marked reduction in IS/AAR (P < 0.05 vs. untreated, Fig. 2). Interestingly, when morphine (0.3 mg/kg) and CCPA (10 µg/kg) were combined (n = 8) and given 10 min before occlusion, no additional protective effect was observed (Fig. 4).
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Blockade of the A1ARs with the selective A1AR antagonist, DPCPX (100 µg/kg), failed to modify IS/AAR compared with untreated hearts (n = 5). When administered 20 min before occlusion, DPCPX completely abolished the cardioprotective effects of both CCPA (n = 6) and morphine (n = 7) (P < 0.05 vs. agonist alone, Fig. 2). Furthermore, pretreatment with DPCPX also eliminated the bradycardic effects of A1AR activation (data not shown).
Interestingly, DPCPX pretreatment produced no change in the protection afforded by the combination of both CCPA and morphine (n = 6, Fig. 4). Even increasing the dose of DPCPX (1 mg/kg, n = 10) failed to attenuate CCPA + morphine-induced cardioprotection (P > 0.05, data not shown).
To examine the effects of DOR inhibition, the selective DOR antagonist BNTX (1 mg/kg) was utilized. Pretreatment with BNTX alone 20 min before ischemia (n = 8) failed to alter IS; however, when given before morphine administration (n = 6), BNTX abolished the protective effect of morphine (P < 0.01 vs. agonist alone, Fig. 2). Surprisingly, BNTX also abolished the protective effect of A1AR activation (n = 7, P < 0.001 vs. agonist alone, Fig. 2). In contrast to DPCPX, pretreatment with BNTX completely blocked protection afforded by the combination of CCPA and morphine (n = 7, P < 0.001 vs. combination alone, Fig. 4).
A3AR inhibition with MRS-1523 (2 mg/kg, n = 7) alone had no effect when given 20 min before occlusion. The dose of MRS-1523 used was determined as the minimum dose required to prevent histamine release from 100 µg/kg Cl-IB-MECA in the intact mouse (Dr. J. Auchampach, personal correspondence). Interestingly, similar to that seen with A1AR blockade with DPCPX, A3AR blockade also inhibited the cardioprotective effects of CCPA (n = 6) and morphine (n = 7), both alone (P < 0.01 and P < 0.05 vs. agonist alone, respectively; Fig. 2) and in combination (n = 6, P < 0.001 vs. combination alone; Fig. 4).
MitoKATP blockade. We examined the role of the mitoKATP channel in mediating the cardioprotective effects of both CCPA and morphine. 5-HD (10 mg/kg) was administered 5 min before drug treatment and 15 min before occlusion. After 90 min of reperfusion, IS/AAR was unaltered by 5-HD treatment alone (n = 6, Fig. 5). However, administration of 5-HD abolished the infarct-limiting effect of CCPA (n = 7) and morphine (n = 6), alone (P < 0.001 and P < 0.01 vs. agonist alone, respectively; Fig. 5) or in combination (n = 9, n = 6, P < 0.001 vs. combination alone; Fig. 4).
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ROS inhibition. The possibility of ROS production as being a trigger of the anti-infarct effects observed with A1AR and DOR activation was investigated with the use of 2-MPG, a free radical scavenger. 2-MPG (20 mg/kg) administered 15 min preocclusion did not alter IS from that of untreated rats (n = 7). When given before treatment with CCPA (n = 7) or morphine (n = 6, n = 8 in combination with CCPA), MPG negated their cardioprotective effects (P < 0.001, P < 0.01, and P < 0.001 vs. agonist/s alone, respectively; Figs. 4 and 5).
The vehicle for MRS-1532 and DPCPX, DMSO, was also administered alone or before morphine treatment, to determine whether the effects of these antagonists were due to the reported free-radical scavenging ability of DMSO. DMSO pretreatment (300 µl/kg, n = 4) did not modify IS in either the untreated rats or rats treated with morphine (n = 4).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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1-opioid antagonist BNTX abolished the
protection afforded by the A1AR agonist CCPA. Although the
mechanism of this interaction is not known, the data clearly suggest that
there is a functional coupling between adenosine and opioid receptors in
mediating cardioprotection in the in vivo rat heart. Furthermore, the data
indicate that both ROS and the mitoKATP channel are involved in
triggering the response to each receptor agonist used. Because adenosine and opioids have both been implicated as endogenous triggers of preconditioning, via A1 and/or A3ARs and DORs (10, 66), and have been shown to modulate similar pathways (10, 23, 34), it is possible that opioid- and adenosine-mediated protective responses may be coupled in the heart as previously suggested for opioids and adrenergic receptors (14) and adenosine and adrenergic receptors (39).
The observed coupling of adenosine and opioid receptors in the CNS (6, 17, 18, 20, 27, 42), coupled with the known cardioprotective effects of adenosine, suggest that opioid receptors and adenosine may act in concert to provide a cardioprotective effect. Indeed, a recent study (28) of the rat heart demonstrated that the cardioprotective effects of fentanyl (a µ-opioid agonist) could be attenuated with an A1AR antagonist and a KATP channel blocker. The results of the present study support the notion that adenosine and DORs act via a converging pathway because no further protection was afforded when agonists of both receptors were applied simultaneously.
Involvement of the mitoKATP channel. The opening of a mitoKATP channel or a mitochondrial site of action appears to be an important trigger for the mediation of cardioprotection. Both ischemic and pharmacological preconditioning may be dependent on the activation of mitoKATP channels; however, the precise role of mitoKATP and sarcolemmal (sarc)KATP channels is controversial. Either KATP channel may trigger cardioprotection (7, 45) or be the end effector (43, 62) or, alternatively, may serve as both (65). The data presented in this study implicate an integral role for the mitoKATP channel in the signal transduction pathway after both acute opioid and adenosine receptor activation. Previous studies (13, 45, 46) from our laboratory support the observation, which suggests that DOR stimulation elicits a cardioprotective effect that is mediated via KATP channels in the intact rat heart. Similarly, Kato et al. (28) demonstrated that the protective effect of fentanyl, an opioid agonist, was blocked by 5-HD. Previous studies in our laboratory (13, 46) have demonstrated that a mitoKATP-dependent mechanism is involved as a distal effector leading to both early and delayed preconditioning conferred by DOR activation in rat hearts.
Of particular interest is our observation that A1AR-mediated protection was also abolished by 5-HD. The role of the KATP channel in mediating protection afforded by adenosine receptor activation is controversial. Whereas several studies support a role for the mitoKATP channel, Grover et al. (16) and Cohen et al. (7) failed to identify an interaction between adenosine receptor activation and KATP channel opening. The report by Cohen et al. (7) may be explained by the differential actions of adenosine itself as opposed to those of selective adenosine receptor agonists (48). In the isolated murine heart, the protective effect of transgenic overexpression of A1ARs was reduced by blockade of the mitoKATP channel (19). Furthermore, we recently reported that cardioprotection mediated via A1AR activation with cyclohexyladenosine is abolished with 5-HD treatment, whereas the protection observed after adenosine administration was not abolished by 5-HD, suggesting that adenosine-mediated cardioprotection is partly independent of mitoKATP opening (48). Indeed, evidence suggests that adenosine may even afford protection independent from that mediated by adenosine receptors (12, 48, 49). Similarly, Patel et al. (47) recently found that some of the cardioprotective effects of a selective DOR agonist are independent of opioid receptor occupation. Thus it is becoming apparent that certain "selective" receptor agonists may have effects independent of their effects on specific receptors and that the A1AR and DOR may be good examples.
Involvement of ROS. Much research has focused on the role of ROS
as a trigger in EPC and DPC. It has been proposed that opening of the
mitoKATP channel leads to a small "burst" of ROS
(31). These oxygen radicals
may then activate/translocate PKC and other intracellular kinases leading to
an unknown effector (7),
possibly the KATP channel. Our observation that opioid-induced
cardioprotection is mediated via ROS is supported by the present results and
the results obtained from previous studies in our laboratory
(47), where the
infarct-limiting effects of a selective -opioid agonist BW-373U86 were
completely abrogated by pretreatment with 2-MPG. Of interest, however, is our
finding with CCPA. This observation is in direct contrast with that of Cohen
and collegues (7), who
demonstrated that protection produced by adenosine could not be inhibited by
2-MPG. Interestingly, Cohen et al.
(7) also reported that
adenosine-mediated protection is independent of mitoKATP channel
opening. However, the opening of the mitoKATP channel appears to
lead to the generation of free radicals
(31) and previous studies
(19,
63) have demonstrated that
adenosine receptor activation mediates protection via downstream
mitoKATP channel opening. Indeed, the results of the current study
indicate that KATP activation is essential for
A1AR-mediated protection. As mentioned earlier, this controversy
may be due to differences between the effects of adenosine itself and
selective adenosine receptor agonists, whereas the cardioprotective effects of
adenosine receptor agonists are dependent on mitoKATP opening,
whereas some effects of adenosine are only partially dependent on
mitoKATP channel opening
(48). Thus activation of the
mitoKATP channel by CCPA may initiate the production or release of
ROS, which then activate downstream kinases leading to myocardial protection.
Collectively, these data demonstrate a pivotal role for the
mitoKATP channel and ROS generation in protection afforded by both
the A1AR and DOR.
Receptor cross-talk. To identify the effects of A1AR and DOR activation in the in vivo rat heart, we studied responses to a selective exogenous A1AR agonist, CCPA and the nonselective opioid agonist, morphine. The data reveal that both A1AR and opioid receptor activation result in a significant reduction in IS. Whereas morphine is a nonselective opioid agonist, it was determined that the protective effects afforded by morphine were attributed to DOR activation because the infarct-limiting effects were abolished with the use of the selective DOR antagonist BNTX. Of particular interest is the observation that the cardioprotection afforded by A1AR or DOR activation could be abolished by BNTX or DPCPX, respectively. Moreover, BNTX failed to antagonize the bradycardic effects of CCPA, suggesting that direct A1AR receptor activation was most likely not being inhibited by BNTX. Whereas A1AR antagonism has previously been shown to abolish opioid-mediated protection in the rat heart (28), these data are the first to suggest that DOR blockade inhibits A1AR-mediated protection. Furthermore, an interaction between opioids and adenosine has been previously documented. Binding of [3H]DPCPX is reduced in the brain of µ-opioid receptor knockout mice (3). DORs are also downregulated in µ-knockout mice (30). Concentrations of cortical A1AR sites are increased after 72 h of morphine treatment in mice (27), and morphine produced a concentration-dependent release of adenosine (51). Moreover, bidirectional cross-withdrawal syndromes were evident when adenosine agonist pretreated rats were administered the opioid antagonist naloxone and when rats pretreated with morphine were given DPCPX or DMPX (8), two adenosine receptor antagonists. Whereas these studies demonstrate a tight coupling between opioids and adenosine in the CNS, the present data suggest a tangible link in cardiac tissue as well.
Simultaneous activation of both the A1AR and DOR failed to enhance the protective effect of either agonist given alone, suggesting a converging pathway or that maximal cardioprotection was produced by either drug at the doses used. This protection was abolished by BNTX, MRS-1523, 2-MPG, and 5-HD, suggesting that this pathway is DOR, A3AR, ROS, and mitoKATP dependent; however, DPCPX failed to blunt the effect of combined A1AR and DOR activation. This observation is of interest because MRS-1523, a selective A3AR antagonist, also blocked the anti-infarct effects of CCPA. This occurred in the absence of any reduction in A1AR-mediated bradycardia, suggesting that there is little or no occupation of the A1AR by MRS-1523. Previous studies in islolated rabbit and rat hearts have demonstrated that A3AR-mediated protection can be attenuated by A1AR antagonists (29), where the affinity of the A3AR for DPCPX was low (35). However, the report by Kilpatrick et al. (29) did not examine the role of the A3AR in A1AR-mediated protection. Shainberg et al. (56) also noted that DPCPX blocked the protection achieved by Cl-IB-MECA and that cardioprotection mediated by CCPA was unaffected by MRS-1523. Whereas the results of Shainberg et al. (56) are in contrast to our findings, this may be explained by differences in the models used. Shainberg et al. (56) used cultured neonatal rat cardiomyocytes, devoid of blood-borne elements, whereas we utilized the in vivo adult rat model. The results of the present study suggest a cross-talk between the A1AR and the A3AR as the A3ARs are resistant to xanthine compounds (35) and the concentration of DPCPX used by Kilpatrick and colleagues (29) was well below that required to occupy A3ARs (35), thus suggesting a relationship between the A1AR and A3AR. Furthermore, overexpression of the A3AR (9) resulted in a reduced basal heart rate, indicating a possible increase in A1AR activation, due to either an increase in receptor number or endogenous adenosine production.
Previous studies (2,
32,
36) have reported that
adenosine is not required for IPC in the rat heart. These observations were
based on pharmacological evidence with A1AR antagonists and 8-SPT,
which may not be inhibiting the A3AR. In addition, CCPA may also
activate the A3AR, as it has been reported that CCPA competes with
[125I]ABA binding with an inhibitor constant 
50 nM to
rabbit-cloned A3ARs
(22). CCPA may activate both
the A1AR, producing both bradycardia and protection, and the A3AR, producing
cardioprotection. Indeed, MRS-1523 abolished the protection afforded by CCPA,
suggesting that either CCPA occupies the A3AR or that activation of the
A1AR leads to activation of the A3AR, further supporting
our hypothesis of receptor cross-talk. Furthermore, Armstrong and Ganote
(1) demonstrated that IPC was
blocked by the nonselective receptor antagonists that occupy A3AR
but not those selective for the A1AR.
Downey et al. (15) proposed that several triggers are required to reach threshold for activation of IPC. According to Downey et al. (15), two or more stimuli may be required to activate PKC to a degree that initiates the signal transduction pathway mediating IPC. Activation of A1/A3ARs and DOR may mediate translocation of PKC (33, 41, 44, 59, 61). Thus when CCPA and morphine are added in combination, the activation of A3ARs via CCPA and enhanced adenosine release mediated by both DOR (18, 51) and A1AR activation (57) may be sufficient to activate PKC to the preconditioning "threshold."
Finally, morphine has been shown to elevate interstitial adenosine levels via an inhibition of adenosine kinase (17). Cyclohexyladenosine increases interstitial adenosine in metabolically stressed cells by a PKC-dependent inhibition of adenosine kinase (57). Moreover, the adenosine kinase sequence has multiple PKC phosphorylation consensus sites (57). Indeed, in the current study, preliminary results obtained by inhibiting adenosine kinase with iodotubercidin (1 mg/kg) afforded similar protection to that provided by either morphine or CCPA (37.5 ± 3.6%, IS/AAR, P < 0.05 vs. untreated). Glibenclamide, a nonselective KATP channel blocker, has been shown to decrease adenosine levels by inhibiting endogenous ecto-5'-nucleotidase, whereas 5-HD has no such effect (52), providing evidence that opening of the sarcKATP channel may mediate an increase in interstitial adenosine. DOR activation also opens sarcKATP channels and triggers delayed preconditioning (45). Thus morphine and CCPA may be enhancing endogenous adenosine concentrations to occupy the A3AR and provide protection via A1AR or A3AR activation and non-receptor-mediated effects (5, 48, 50). Furthermore, adenosine deaminase predominates as the pathway for adenosine metabolism at times when adenosine levels are high (21, 25). Increased activation of adenosine deaminase during adenosine kinase inhibition will result in increased levels of inosine. Inosine has been shown to bind to A3ARs at concentrations between 10 and 50 µM (24, 60).
In conclusion, these data reveal that A1AR and DOR agonists mediate cardioprotection via a mitoKATP and ROS-dependent mechanism. Our observations suggest that the A3AR is intimately involved in both A1AR- and DOR-mediated protection. Furthermore, there appears to be convergence between the pathways linking A1AR- and DOR-mediated protection. Indeed, it appears that A1AR and DOR activation may act in concert to afford cardioprotection.
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ACKNOWLEDGMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-08311 (to G. J. Gross) and a Postdoctoral Fellowship from the American Heart Association, Northland Affiliate (to J. N. Peart).
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FOOTNOTES |
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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.
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H. Kin, A. J. Zatta, M. T. Lofye, B. S. Amerson, M. E. Halkos, F. Kerendi, Z.-Q. Zhao, R. A. Guyton, J. P. Headrick, and J. Vinten-Johansen Postconditioning reduces infarct size via adenosine receptor activation by endogenous adenosine Cardiovasc Res, July 1, 2005; 67(1): 124 - 133. [Abstract] [Full Text] [PDF]
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H. H. Patel, E. R. Gross, J. N. Peart, A. K. Hsu, and G. J. Gross Sarcolemmal KATP channel triggers delayed ischemic preconditioning in rats Am J Physiol Heart Circ Physiol, January 1, 2005; 288(1): H445 - H447. [Abstract] [Full Text] [PDF]
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G. Kristo, Y. Yoshimura, B. J. Keith, R. M. Stevens, S. A. Jahania, R. M. Mentzer Jr., and R. D. Lasley Adenosine A1/A2a receptor agonist AMP-579 induces acute and delayed preconditioning against in vivo myocardial stunning Am J Physiol Heart Circ Physiol, December 1, 2004; 287(6): H2746 - H2753. [Abstract] [Full Text] [PDF]
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S. M. White, P. E. Constantin, and W. C. Claycomb Cardiac physiology at the cellular level: use of cultured HL-1 cardiomyocytes for studies of cardiac muscle cell structure and function Am J Physiol Heart Circ Physiol, March 1, 2004; 286(3): H823 - H829. [Abstract] [Full Text] [PDF]
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P < 0.05 vs. agonist alone.
