Cardiovascular Research

Cardiovascular Research 2002 55(3):429-437; doi:10.1016/S0008-6363(02)00439-X
© 2002 by European Society of Cardiology

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Copyright © 2002, European Society of Cardiology

Mitochondrial K_ATP channels: role in cardioprotection

Olaf Oldenburg^a, Michael V Cohen^a,b, Derek M Yellon^c and James M Downey^a,*

^aDepartment of Physiology, MSB 3024, University of South Alabama, College of Medicine, Mobile, AL 36688, USA
^bDepartment of Medicine, MSB 3024, University of South Alabama, College of Medicine, Mobile, AL 36688, USA
^cThe Hatter Institute and Center for Cardiology, University College Hospital and Medical School, Grafton Way, London WC 1E 6DB, UK

jdowney{at}usouthal.edu

^* Corresponding author. Tel.: +1-251-460-6818; fax: +1-251-460-6464

Received 10 December 2001; accepted 9 April 2002

	Abstract

Top Abstract 1. Is the mKATP... 2. Can mKATP act... 3. mKATP and downstream... 4. mKATP and reactive... 5. Studies using A7r5... 6. Receptors that couple... 7. What is the... 8. Conclusion References

 
The role of the mitochondrial ATP-sensitive potassium channel (mK_ATP) in ischemic preconditioning and cardioprotection is reviewed. A great deal of accumulated evidence implicatese opening of this channel as an important step in the anti-infarct effect of ischemic preconditioning. Recent studies, however, reveal that channel opening can actually serve as a signal transduction element. Data indicate that mK_ATP opening causes mitochondria to generate reactive oxygen species (ROS) which then activate downstream kinases. Opening of mK_ATP prior to ischemia can serve as a trigger since the critical time for its opening is prior to the onset of the lethal ischemic insult. Most G_i-coupled receptors trigger protection through the mK_ATP/ROS pathway except for the adenosine receptor which uses some other, as yet unidentified, pathway. Possible coupling schemes between the receptors and the mK_ATP are discussed. Protection from preconditioning can also be aborted when a mK_ATP blocker is present only during the lethal ischemic insult (mediator phase), but a much higher concentration of the blocker is required. Thus the mK_ATP probably serves a dual role as both a trigger and a mediator. Possible end-effectors of preconditioning's protection are discussed including the mK_ATP itself.

KEYWORDS Free radicals; K-ATP channel; Mitochondria; Preconditioning; Signal transduction

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Ischemic preconditioning (PC) is a phenomenon whereby one or more brief episodes of ischemia protect the heart against a subsequent lethal ischemic insult. There is first an early phase of protection that lasts for 1–2 h after the brief preconditioning ischemia in anesthetized animals [1–4] and more than 2 but less than 4 h in conscious rabbits [5]. A second window of protection [6] appears about 12–24 h after the preconditioning event and lasts several days. After more than a decade since this phenomenon was first described in dog hearts [7] some of the underlying pathways have been resolved, but the end-effector of this protection has remained elusive. While there is good agreement that the mitochondrial ATP-sensitive potassium channel (mK_ATP) plays a major role in ischemic preconditioning, controversy exists as to whether this channel is a trigger or mediator of preconditioning or even the end-effector. Moreover, the exact role reactive oxygen species (ROS) play in preconditioning and their source have also not been clear despite unequivocal documentation of their involvement for several years [8,9]. In this review we will examine the accumulating evidence that the mK_ATP channel acts as a trigger of ischemic preconditioning, and that channel opening causes the generation of ROS which become important signals in the intracellular pathway responsible for preconditioning's protection.

	1. Is the mKATP channel involved in preconditioning?

Top Abstract 1. Is the mKATP... 2. Can mKATP act... 3. mKATP and downstream... 4. mKATP and reactive... 5. Studies using A7r5... 6. Receptors that couple... 7. What is the... 8. Conclusion References

 
The K_ATP channels were first linked to preconditioning by Gross et al. [10]. Blocking the channel with glibenclamide in dog hearts prevented preconditioning's protection, while the drug had no effect on the non-preconditioned heart. Although glibenclamide closes both mitochondrial and sarcolemmal K_ATP channels, it was initially assumed that the sarcolemmal K_ATP was the end-effector of PC's protection, and this protection was originally ascribed to shortening of the action potential (see Ref. [11] for review). At the time of these early observations it was unknown that cardiomyocytes contained two different types of K_ATP channels, sarcolemmal and mitochondrial, and that each had a distinct pharmacological profile. However, Garlid et al. [12], Liu et al. [13] and Sato et al. [14] subsequently provided convincing evidence in different models that it was indeed not the surface but the mK_ATP channel that was involved in protection. Although some data, particularly studies using HMR 1098 [15], support a critical role for the sarcolemmal K_ATP channel, most evidence is consistent with a role for the mitochondrial, rather than the surface, channel. However, because it has not been easy to explain how opening of mK_ATP should be protective, some have questioned whether these mitochondrial channels could be end-effectors.

The mitochondria make ATP by allowing H⁺ extruded by the electron transport apparatus to reenter along a strong electrochemical gradient through the F1 apparatus. In so doing ADP is phosphorylated to ATP. Opening the K_ATP channel will cause potassium to enter mitochondria along its favorable electrochemical gradient. The potassium–hydrogen exchanger on the inner mitochondrial membrane allows intramitochondrial potassium to exchange for extramitochondrial H⁺. Entering H⁺ uncouples the mitochondrion because it bypasses F1 and hence reduces ATP production. In actuality, however, the amount of uncoupling resulting from potassium entry is very small (estimated to be approximately 5 mV), assumed to be caused by a low density of channels in the inner membrane [16]. Terzic's group has reported the greatest change which was 10 mV with a baseline of –180 mV [17]. The potassium that enters is, however, osmotically active and will cause the matrix to swell.

There are several theories that seek to explain why opening the mK_ATP should be protective. Terzic and co-workers found that opening mK_ATP made isolated mitochondria more resistant to Ca²⁺ entry [18]. Garlid [19] suggests that mitochondrial swelling subsequent to potassium entry causes preservation of the functional coupling between mitochondrial creatine kinase and adenine nucleotide translocase on the outer membrane through which ADP traditionally enters the intermembrane space. That juxtaposition effectively keeps ADP out of the intermembrane space and forces the mitochondria to phosphorylate only creatine which is the most efficient means of transferring energy to the cytoplasm.

	2. Can mKATP act as a trigger of preconditioning?

Top Abstract 1. Is the mKATP... 2. Can mKATP act... 3. mKATP and downstream... 4. mKATP and reactive... 5. Studies using A7r5... 6. Receptors that couple... 7. What is the... 8. Conclusion References

 
Recent experiments have re-examined the assumption that mK_ATP is only an end-effector of protection. Liu et al. [13] introduced a cardiomyocyte model in which FADH fluorescence is monitored. The slight uncoupling with mK_ATP opening was proposed to slightly oxidize the flavoproteins and increase their fluorescence. These fluorescence studies showed that the effects of both diazoxide, an mK_ATP opener, and 5-hydroxydecanoate (5HD), a putative selective mK_ATP closer, are readily reversible when the drugs are washed out. Yet Ashraf's group [20] reported that a 5-min pulse of diazoxide followed by washout put the rat heart into a preconditioned state even though the mK_ATP channels should have closed when diazoxide was washed out. Pain et al. [21] repeated the experiment in the isolated rabbit heart and found that a 5-min pulse of diazoxide protected the heart and that the drug could be washed out for as long as 30 min without loss of the protective effect as shown in Fig. 1. Pinacidil, a non-selective K_ATP opener, had the same effect. These data suggested that opening mK_ATP triggered protection and caused the cell to remember that the channel had been open long after it should have closed again.

View larger version (20K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Infarct size as a percentage of the risk zone. The isolated rabbit heart remains protected against a sustained episode of ischemia (30 min), even when the KATP channel opener (either 10 µM diazoxide or 100 µM pinacidil) is allowed to wash out for 15 min (diazoxide, pinacidil) or 30 min (diazoxide). The times indicated beneath the abscissa specify the duration of reperfusion or drug washout. () Individual infarct size, () group mean±S.E.M. *P<0.05 versus control. PC, ischemic preconditioning. Adapted from Ref. [21].

 
Pain et al. [21] further studied the timing of channel opening required to protect ischemic hearts. They set out to determine whether mK_ATP opening was needed to trigger or mediate protection. This distinction is important and has helped to establish the order of cellular events in the signal transduction pathway leading to protection. For example, if an adenosine receptor blocker were given early to bracket the brief ischemic period of a preconditioning protocol and were then washed out prior to the prolonged ischemia, it would block PC's protection revealing that adenosine is a trigger [22]. On the other hand, a PKC inhibitor has no effect against protection when given early, but completely blocks PC's protection when given late just before and during the prolonged ischemia [23], thus identifying PKC as a mediator rather than trigger of protection. Triggers act prior to the index ischemia and are presumably upstream events, while mediators act during the index ischemia and therefore must be downstream events. The end-effector of preconditioning would be expected to fall into the mediator category, but it is important to realize that a mediator is not necessarily the end-effector of protection.

To investigate whether mK_ATP is a trigger or mediator Pain et al. [21] and Wang et al. [24] used isolated rabbit hearts and two schedules for administration of 5HD. To test whether mK_ATP channels act as triggers of preconditioning, both Pain and Wang used a schedule in which 5HD was administered early to bracket the preconditioning ischemia followed by a period of drug washout prior to the long ischemia. In both studies ‘early’ 5HD blocked protection supporting a trigger role (Fig. 2). While Pain et al. [21] were unable to block protection with 5HD given in the late protocol just prior to and during the long ischemia, Wang et al. [24] could abort protection when the concentration of 5HD was increased 4-fold over that required to prevent protection in the early protocol. They proposed that a higher concentration was required because channel phosphorylation reduced the potency of 5HD for channel blockade [25]. Thus while both investigative groups agree that mK_ATP can act as a trigger of protection, Wang proposed that these channels are mediators as well.

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Protection by ischemic preconditioning (pt) induced in the isolated rabbit heart requires opening of the mKATP only during the trigger phase. 5HD (E), (L), 5-hydroxydecanoate administered either early (E) or late (L) as depicted in the inset; () individual infarct size; () group mean±S.E.M. *P<0.05 versus control. Adapted from Ref. [21].

 
Further support for mK_ATP as a mediator comes from Gross et al. [10] who infused 5HD in dogs between the time of PC and the index ischemia and blocked protection. Yao et al. [26] also noted in their chick cell model that protection from a PKC activator could be blocked when 5HD was introduced only during prolonged simulated ischemia. Thus the current weight of evidence supports both a trigger and a mediator role for the channel. An attractive explanation of this dual role would be a scenario in which channel opening triggers a kinase cascade that feeds back in a positive manner to keep the channel open during the index ischemia.

	3. mKATP and downstream kinases

Top Abstract 1. Is the mKATP... 2. Can mKATP act... 3. mKATP and downstream... 4. mKATP and reactive... 5. Studies using A7r5... 6. Receptors that couple... 7. What is the... 8. Conclusion References

 
If mK_ATP opening is an upstream event, then where in the pathway are the signaling kinases located? Ashraf's group [20] showed that the PKC blocker chelerythrine could block protection from a pulse of diazoxide in the isolated rat heart. While Pain et al. [21] could not show a similar result in the rabbit heart, they were able to block diazoxide's protection with the tyrosine kinase blocker genistein, indicating that there was at least one tyrosine kinase downstream from mK_ATP opening in the rabbit model. Thus mK_ATP opening protects by activating kinases. We interpret this to further indicate that mK_ATP opening can act as an upstream link in a signal transduction chain. This proposal will be examined in more detail later in this review.

	4. mKATP and reactive oxygen species

Top Abstract 1. Is the mKATP... 2. Can mKATP act... 3. mKATP and downstream... 4. mKATP and reactive... 5. Studies using A7r5... 6. Receptors that couple... 7. What is the... 8. Conclusion References

 
The final piece of this puzzle was provided by Steenbergen and co-workers. They found that diazoxide's protection could be blocked by a free radical scavenger, N-acetylcysteine [27]. Their observation was then confirmed by Pain et al. [21] who used the scavenger N-2-(mercaptopropionyl) glycine (MPG). Furthermore Yao et al. [28] found that pharmacological preconditioning of chick cardiomyocytes with the muscarinic agonist acetylcholine (ACh) caused the cells to produce a small burst of free radicals as shown in Fig. 3. Yao et al. used the probe 2',7'-dichlorofluorescein diacetate (DCFH) which fluoresces when oxidized by free radicals. This burst could be blocked by myxothiazol [29,30], indicating that the increased ROS production was the result of electron transport within the mitochondria, probably from site III of the electron transport chain where myxothiazol blocks the flow of electrons (for review of free radicals and their cellular origins see Ref. [31]). Also the burst could be blocked by 5HD. These observations led Pain et al. [21] to propose a new paradigm incorporating mK_ATP and ROS in PC's signaling pathway leading to protection (Fig. 4). In this model receptor occupancy leads to mK_ATP channel opening which then causes the mitochondria to produce ROS. The free radicals would then activate the downstream kinases that ultimately modulate the end-effector.

View larger version (26K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Acetylcholine (ACh) administration caused free radical formation in chick cardiomyocytes (A). That burst could be blocked by myxothiazol (MYXO) (B), N-2-(mercaptopropionyl) glycine (MPG) (C) or 5-hydroxydecanoate (5HD) (D). CONT, control. Reprinted with permission from Ref. [28].

 

View larger version (48K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Proposed signaling pathway for receptor-mediated protection. Gi-protein-linked agonists bind to a surface receptor. For receptors other than adenosine subsequent steps include activation of phosphatidylinositol 3 (PI3)-kinase, at least one tyrosine kinase and possible other steps to open mitochondrial KATP channels. The linked release of reactive oxygen species (ROS), possibly in context with other mediators, leads to activation of protein kinase C (PKC) and other tyrosine kinases. The purpose of all these steps is to activate the end-effector, which then mediates protection.

 
It is unclear why opening the mK_ATP channel would cause ROS production. Garlid (personal communication) has speculated that by extruding H⁺ potassium entry into mitochondria has the effect of alkalinizing the matrix, and alkalinization is known to promote ROS production by the electron transport proteins.

It is interesting that free radicals have been known for some time to be involved in PC. Both Baines et al. [9] and Tritto et al. [8] found that free radical scavengers could block PC's protection and that a free radical generating system could duplicate it. Both groups, however, concluded that the radicals were simply a consequence of the preconditioning ischemia–reperfusion and were working in concert with surface receptors to activate PKC. The present data suggest that those radicals play a much more central role and that they are actually coming from the mitochondria under the control of mK_ATP as illustrated in Fig. 4. While this model may not explain all of the available data, we believe that it comes the closest.

Several studies now confirm that opening mK_ATP channels results in ROS production in cells. As already noted Yao et al. [28] demonstrated that the increase in ROS production induced by ACh could be blocked by 5HD. More recently Yellon's group [32] examined cells derived from human atria and treated them with diazoxide. Free radical production was monitored by loading the cells with reduced MitoTracker^® Orange which becomes fluorescent only after oxidation by ROS. The oxidized species is then trapped by covalent binding to proteins within the mitochondria making the cells fluoresce. They found that treatment with diazoxide causes an increase in fluorescence. The possible impact of diazoxide and ROS on the mitochondrial membrane potential remains controversial. Using the voltage-sensitive dye JC-1 Yellon [32] found no change in mitochondrial membrane potential, whereas Murata et al. [33] used confocal microscopy to conclude that diazoxide resulted in a 12% change in mitochondrial membrane potential. The latter observation is supported by Terzic's data of a similar K⁺ channel opener-induced membrane depolarization [17]. It is unknown whether these changes in potential are directly linked to the release of free radicals or changes of Ca²⁺ homeostasis. Clearly more work is needed in this area. The problem is compounded by the fact that most mK_ATP openers are also non-specific mitochondrial uncouplers that can reduce membrane potential independent of the mK_ATP. Murata et al. [33] found that diazoxide's effect on membrane potential was concentration-dependent, but that effects of concentrations of diazoxide exceeding 10 µM could not be inhibited by 5HD indicating a non-specific uncoupling. Most studies of PC have used concentrations beyond that range.

Forbes et al. [27] used DCFH to monitor ROS production in rat cardiomyocytes. Diazoxide did cause a slight but significant increase in ROS production in these cells. On the other hand Liu et al. [34] used similar methodology in rabbit cardiomyocytes and failed to see any increase in fluorescence after exposure of the cells to diazoxide. Our own experience is similar, and we found that MitoTracker^® was a more sensitive probe for detection of diazoxide-induced ROS formation.

	5. Studies using A7r5 cells

Top Abstract 1. Is the mKATP... 2. Can mKATP act... 3. mKATP and downstream... 4. mKATP and reactive... 5. Studies using A7r5... 6. Receptors that couple... 7. What is the... 8. Conclusion References

 
Krenz et al. [30] recently developed a cellular model in A7r5 vascular smooth muscle cells that has enabled us to further dissect the role of mitochondria and ROS in the signal transduction pathway leading to protection. The line of immortal smooth muscle cells was chosen for these experiments for practical reasons to eliminate the labor-intensive step of isolating fresh cardiomyocytes on a daily basis. In this model cells are loaded with reduced MitoTracker^® Red (identical to MitoTracker^® Orange except fluorescence is red instead of orange). Diazoxide increased fluorescence dramatically and that increase could be blocked by 5HD supporting K_ATP involvement. The acknowledged problem with a non-cardiomyocyte cell type such as that used by Krenz et al. [30] and Carroll et al. [32] is that the specificity of diazoxide and 5HD for the mitochondrial over sarcolemmal K_ATP has yet to be determined. Thus, while it is safe to assume that these agents are specific for some type of K_ATP channel, we cannot say with certainty that they are exclusively opening and closing mitochondrial channels. It should be pointed out, however, that diazoxide and 5HD modulated ROS production in our A7r5 cells in a manner identical to that seen in the two described studies which examined cardiomyocyte responses [27,28], thus increasing the likelihood that mK_ATP is the target of diazoxide and 5HD in the vascular smooth muscle cells.

The increase in fluorescence induced by diazoxide in the A7r5 cells could be blocked by myxothiazol, an electron transport blocker, proving a mitochondrial origin of at least some of the ROS. The free radical scavenger MPG also blocked the increased fluorescence indicating that it was ROS that caused the change in fluorescence rather than an alteration in mitochondrial membrane potential. The ROS production is apparently a direct result of potassium influx into the mitochondria because the potassium ionophore valinomycin caused an increase in fluorescence similar to that of diazoxide [35]. Interestingly, valinomycin's increase in fluorescence could not be blocked with 5HD which would be expected since valinomycin does not use the K_ATP to promote potassium movement. Hence K⁺ movement, presumably into mitochondria, appears to lead to production of ROS, and the latter seem to be important signaling elements.

The MitoTracker^® probe is concentrated in mitochondria which we believe greatly increases its sensitivity to mitochondrial-derived ROS. A potential shortcoming and criticism of the MitoTracker^® probe is that its concentration in the mitochondria is quite voltage-dependent [36]. In an attempt to remove the voltage-dependent pool we washed the cells several times at the end of our incubation period before measuring fluorescence. This was intended to create a strong concentration gradient favoring washout of unbound probe from the mitochondria. After washout we found that the level of fluorescence remained constant for over 30 min, suggesting that most of the unbound MitoTracker^® had washed out. To be sure that changes in fluorescence were related to increased ROS production rather than an alteration in mitochondrial membrane potential, we repeated our studies with oxidized MitoTracker^® which is fully fluorescent and insensitive to ROS [30] so that changes in fluorescence would indicate changes in total amount of probe rather than only the amount of oxidized probe which is dependent upon exposure to ROS. In those experiments diazoxide never increased fluorescence, proving that voltage effect could not explain the increased fluorescence. More importantly depolarization of the mitochondria with DNP had no effect on the oxidized MitoTracker^® fluorescence, indicating that the MitoTracker^® uptake as used in our protocol is surprisingly voltage-independent.

It is well known that surface receptors such as bradykinin, -adrenergic, and muscarinic can trigger PC, and recent data suggest that these receptors are linked to mK_ATP channels and free radicals (Fig. 5) [37]. To gain further insight into these very early signaling steps A7r5 cells which have intact muscarinic receptors were exposed to ACh. As presented in this issue of Cardiovascular Research [38] we now have resolved some of the signaling pathway by which ACh leads to ROS generation, at least in vascular smooth muscle cells. ACh caused an increase in fluorescence that was not different from that generated by diazoxide. That increase could be blocked with muscarinic receptor antagonists including atropine, proving that it was receptor-mediated. Muscarinic receptors are coupled to G_i and, as expected, pertussis toxin blocked the response to ACh. Pertussis toxin did not affect the increase in fluorescence from either diazoxide or valinomycin, proving that the mitochondria were responding normally in the presence of the toxin. Because G proteins are confined to the cell membrane, we reasoned that there must be some way of coupling these G proteins to a cytoplasmic pathway that would then be able to interact with mitochondria. The phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin blocked ACh's ability to increase cell fluorescence, indicating that 3-phosphoinositides carry the signal to the interior of the cell (assuming that mK_ATP and not surface channels are the target). We were also able to block the ACh response with genistein, suggesting that there is also at least one tyrosine kinase in this pathway. Neither wortmannin nor genistein could block the increased fluorescence from diazoxide, again showing that neither agent had affected the mitochondria directly, and that involvement of PI3-K and tyrosine kinase in this signaling are upstream of mK_ATP channels (Fig. 4). Indeed Tong et al. [39] found that wortmannin blocked protection from preconditioning at a very upstream site which correlates quite well with our findings in A7r5 cells.

View larger version (15K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Acetylcholine (ACh) mimics ischemic preconditioning in the isolated rabbit heart. Its protection is blocked during the trigger phase by either the mKATP channel closer 5-hydroxydecanoate (5HD, 200 µM) or the free radical scavenger N-(2-mercaptopropionyl) glycine (MPG, 300 µM). () Individual infarct size; () group mean±S.E.M. *P<0.01 versus ACh. Adapted from Ref. [37].

 
We propose that opening of K_ATP leads to production of free radicals (presumably by mitochondria since myxothiazol blocked it). Protein kinase C (PKC) is intimately involved in preconditioning's protection [4]. Wang et al. [20] found that blocking PKC also abrogated diazoxide's ability to precondition the rat heart. This would seem to suggest that PKC is downstream of mK_ATP, and would argue for a trigger role of the mK_ATP. Because of the authors’ assumption that mK_ATP channels are the end-effectors rather than triggers, they circumvented this conundrum by proposing that diazoxide opens the mK_ATP channel by first activating PKC [20]. To test that hypothesis, Krenz et al. [30] tried to block the ROS signal from diazoxide with the PKC blocker chelerythrine. Chelerythrine did not block the diazoxide-induced increase in fluorescence, strongly refuting Wang's conclusion. Furthermore, it should be pointed out that PKC would not have been present in the mK_ATP-containing liposomes in which Garlid et al. first showed that diazoxide opens the mK_ATP [40]. Thus we interpret Wang's data to show PKC must be downstream of K_ATP and presumably of ROS production.

	6. Receptors that couple to the mKATP

Top Abstract 1. Is the mKATP... 2. Can mKATP act... 3. mKATP and downstream... 4. mKATP and reactive... 5. Studies using A7r5... 6. Receptors that couple... 7. What is the... 8. Conclusion References

 
Liu et al. [41] first discovered that preconditioning was receptor-mediated. They found that adenosine occupied A₁ receptors to trigger the preconditioned state. It is now known that adenosine receptors do not act alone in PC, but, at least in the rabbit, participate in concert with both bradykinin [42,43] and opioid receptors [44]. Ligands to all three receptor types are released during ischemia (endogenous agonists) and precondition the heart by a PKC-dependent mechanism [4]. Blocking one receptor type does not completely prevent protection, but rather only raises the ischemic threshold required to put the heart into a PC state so that increased activation of the remaining receptor types is necessary to successfully trigger protection. Furthermore it can be shown that virtually all G_i-coupled receptors present on the cardiomyocyte are capable of preconditioning the myocardium, even if their ligands (exogenous agonists) are not released during a PC protocol. These ‘other’ receptors include the muscarinic M₂ [45], angiotensin AT₁ [46], adrenergic ₁ [47], and endothelin ET₁ [48].

Any receptor signaling through the mitochondrial pathway should therefore be blocked by either closing the mK_ATP channel with 5HD or by intercepting the released reactive oxygen species with MPG. Using the Langendorff rabbit heart model, Cohen et al. [37] tested this hypothesis. Every ligand was administered at approximately 10 times its known K_d for 5 min and washed out for 10 min prior to the index ischemia. MPG (300 µM) or 5HD (200 µM) was given to bracket the ligand pulse (the early schedule as detailed above). Fig. 5 shows that both MPG and 5HD blocked protection triggered by the muscarinic agonist ACh, indicating involvement of both mK_ATP and free radicals. Similar blockade of protection by MPG and 5HD was found when opioid receptors were activated by morphine, bradykinin receptors by bradykinin, and -adrenergic receptors by phenylephrine. To the authors’ surprise, however, adenosine or the A₁-selective adenosine agonist N⁶-(2-phenylisopropyl) adenosine (PIA) behaved very differently. Neither 5HD nor MPG could block their protection. This indicated that adenosine uses a very different signaling pathway to precondition the heart than the other receptors. The reason for the difference is not clear as adenosine A₁ and muscarinic receptors were always thought to share the same G_i protein. Despite the inability to explain why this divergence exists, it is apparent that there are two parallel signaling pathways. It is interesting that the signaling of preconditioning is quite redundant, and this redundancy is seen at multiple levels. There are at least three surface receptors acting in parallel which can trigger preconditioning. We also know that there are at least two parallel kinase pathways. The protection of ischemic preconditioning in the pig could not be blocked with either the PKC antagonist staurosporine or the tyrosine kinase blocker genistein when each was administered singly, and was successfully abrogated only by co-administration of the two agents [49]. Furthermore, increasing the number of cycles of preconditioning in the rat will overcome either PKC or tyrosine kinase blockade but not the two in combination [50,51]. Now we find that there are two parallel pathways between the receptors and kinases. This means that there is no single point (other than perhaps the end-effector) where one can absolutely block preconditioning's protection with a single intervention.

	7. What is the end-effector of PC's protection?

Top Abstract 1. Is the mKATP... 2. Can mKATP act... 3. mKATP and downstream... 4. mKATP and reactive... 5. Studies using A7r5... 6. Receptors that couple... 7. What is the... 8. Conclusion References

 
Activators of the mediator phase of preconditioning must act at downstream sites either shortly before and/or during the index ischemia. Although the end-effector(s) of protection must be a mediator, not all mediators will be end-effectors. As described above, mK_ATP clearly acts as a trigger, and several investigators have concluded that the channels may also be mediators.

Is mK_ATP the end-effector? Several scenarios have been discussed above to explain how mK_ATP opening could have a direct beneficial effect. Nevertheless these channels could still be acting as signal transduction elements even during the mediator phase. However, if the mK_ATP is only a signal transduction element in PC, then what effects the protection? The short answer is that we do not know. We have assumed the mK_ATP was the end-effector for so long that few alternatives have been considered. Anion channels have been proposed [52], but our measurements could not confirm this hypothesis [53]. The cytoskeleton [54], particularly actin filaments [55], has also been suggested to be the end-effector. Murry et al. [56] considered a direct metabolic effect a decade ago, but it is not always possible to correlate protection with preservation of high-energy phosphates [57]. Elsewhere in this issue Garcia-Dorado et al. [58] present compelling evidence that closure of gap junctions may play a role.

	8. Conclusion

Top Abstract 1. Is the mKATP... 2. Can mKATP act... 3. mKATP and downstream... 4. mKATP and reactive... 5. Studies using A7r5... 6. Receptors that couple... 7. What is the... 8. Conclusion References

 
The mitochondrial K_ATP channels play an important role in cardioprotection. This review has focused on the compelling evidence that they can act as signal transduction elements to trigger as well as mediate the PC state.

Time for primary review 21 days.

	References

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