POSTCONDITIONING, repetitive ischemia applied before reperfusion, protects against ischemia–reperfusion injury.1Of the therapies proposed for protecting ischemic myocardium, postconditioning offers a significant clinical advantage; it obviates predicting when someone will have an ischemic attack. As such, mechanisms involved in postconditioning are of significant interest. In this issue of Anesthesiology, Jang et al.  2demonstrate that ischemic postconditioning in the heart involves activation of δ-opioid receptors. Morphine, a mixed opioid agonist, produced postconditioning that was abolished by a δ-opioid receptor antagonist or pharmacologic opening (via  atractyloside) of the mitochondrial permeability transition pore (mPTP). The authors showed that morphine exposure in isolated cardiac myocytes produced nitric oxide and attenuated hydrogen peroxide oxidant stress–induced loss of the mitochondrial membrane potential (ΔΨm). The attenuation of ΔΨmproduced by morphine was sensitive to δ-opioid antagonism, a nonselective nitric oxide synthase inhibitor and an inhibitor of protein kinase G. The authors concluded that (1) postconditioning protects the heart by targeting the mPTP via  activation of δ-opioid receptors and (2) the ability of δ opioids to activate the nitric oxide–cyclic guanosine monophosphate–protein kinase G pathway may account for the effect of postconditioning on the mPTP. The authors are to be complimented for their original work regarding a role for δ-opioid signaling on the mPTP.

Mitochondria, a source of cellular adenosine triphosphate, are increasingly being implicated in cell survival and death signaling. mPTP opening leads to an increase in mitochondrial membrane permeability to small molecules and plays an integral role in regulating cytoprotection. The mPTP, a putative high conductance channel on the inner mitochondrial membrane, is thought to be the final end effector in cardiac myocyte protection and therefore an important therapeutic target for cardiac protective strategies.3,4The molecular composition of the mPTP is controversial. The pore putatively is composed of the adenine nucleotide translocase on the inner mitochondrial membrane, voltage-dependent anion channel on the outer membrane, and cyclophilin D in the mitochondrial matrix. These components exist as individual entities that assemble into a complex in response to stress to form the mPTP.3A benzodiazepine receptor, hexokinase, and creatine kinase also have been proposed as regulators of the pore. The role of adenine nucleotide translocase and voltage-dependent anion channel in forming the pore recently has been questioned. The adenine nucleotide translocase may act as a regulator of the mPTP pore, but not as a pore-forming unit of the complex.5In addition, voltage-dependent anion channel knockout mice (voltage-dependent anion channels 1, 2, 3, 1/3, and 1/2/3) show stress-induced mPTP opening indistinguishable from wild-type mitochondria, questioning whether the voltage-dependent anion channel is an essential component of the pore.6Cyclophilin D seems to be the only essential component of the mPTP described thus far.7The authors treat the mPTP as one entity, not a multiprotein complex. It is unclear whether cardiac protective agents work to inhibit the opening of a preformed mPTP complex, inhibit a particular subunit of the complex, or inhibit the assembly or organization of the complex. Recent work showed that increased phosphorylation of glycogen synthase kinase-3β in a model of protection reduces the affinity of the adenine nucleotide translocase for cyclophilin D, suggesting that assembly of the complex is targeted by protective signals to limit mPTP opening.8 

A limitation of the current study deals with the indirect means by which mPTP opening was assessed. Tetramethylrhodamine ethyl ester, a dye used to measure ΔΨm, was used to infer mPTP opening. Although the literature largely agrees that mPTP opening can be inferred by loss of ΔΨm, they are not one in the same. A loss in ΔΨmcan be caused by factors other than mPTP opening (e.g. , increased adenosine triphosphate demand). To circumvent this limitation, calcein acetoxymethylester–loaded cells in the presence of Co2+can be used.9Calcein is loaded into cells and taken up by mitochondria. Residual calcein is quenched by Co2+. If a stress is induced to open the mPTP, calcein is released and fluorescence is lost; this is reversed by addition of cyclosporine A. Dual loading of cells with tetramethylrhodamine ethyl ester and calcein can measure both ΔΨmand mPTP opening simultaneously.10In the whole heart, mPTP opening can be assessed by a method devised by Halestrap et al.  11,12in which radioactive 2-deoxyglucose (3H-DOG) is loaded into cells and accumulates as a phosphate. Functioning mitochondria exclude the 3H-DOG, and opening of the mPTP allows accumulation of 3H-DOG in mitochondria, which can be assessed by isolating mitochondria in the presence of EGTA to trap mitochondrial 3H-DOG during isolation.

The authors also used atractyloside, a pharmacologic mPTP opener, to block ischemic and opioid postconditioning, leading to the conclusion that opioids impact mPTP opening to affect postconditioning. Because mPTP opening is proposed to be an end effector of protection, interventions that open the mPTP pharmacologically would be expected to abrogate cardiac protection induced by any form of preconditioning or postconditioning (i.e. , ischemic or pharmacologic). Atractyloside would not implicate a role for protective agents in the modulation of mPTP. Therefore, use of atractyloside does not address the role of mPTP in protection, but shows that mPTP opening is responsible for tissue injury. In addition, it has been reported that atractyloside not only opens mPTP but also inhibits adenosine diphosphate transport by inhibition of adenine nucleotide translocase,13therefore limiting oxidative phosphorylation. As such, it would be difficult to differentiate the effects of atractyloside on mPTP opening versus  loss of energy production as causative factors in attenuated protection. Therefore, to assess whether protective agents use mPTP as a downstream mechanism, future studies should directly test whether these agents impact mPTP opening in response to stress.

The role of nitric oxide and/or reactive oxygen species in postconditioning events is intriguing, especially with respect to their impact on the mPTP. We and others have shown a role for reactive oxygen species in triggering postconditioning induced by ischemia, isoflurane, and the δ-opioid agonist SNC-121.14,15There is evidence that reactive oxygen species may impact downstream mediators of protection (e.g. , mitochondrial adenosine triphosphate–sensitive potassium channels)16; however, the nature of the reactive species generated and the role in inducing protection is under debate. During severe ischemia–reperfusion injury, overproduction of reactive oxygen species and mitochondrial Ca2+overload produce mPTP opening, ΔΨmdepolarization, inhibition of adenosine triphosphate production, mitochondrial swelling, additional reactive oxygen species production, and further Ca2+accumulation, all of which initiate mitochondrial dysfunction. However, reactive oxygen species at low concentrations can initiate signaling cascades that preserve mitochondrial integrity and myocardium during ischemia and reperfusion. The finding by Jang et al.  2that morphine generates nitric oxide and that changes in ΔΨmare sensitive to nitric oxide synthase inhibition may provide a potential mechanism of action for opioids in ischemic postconditioning and implicate nitric oxide as the triggering reactive species. Low levels of endogenous nitric oxide and low concentrations of nitric oxide donors (<1 μm) protect mitochondria via  suppression of mPTP opening, whereas high concentrations of nitric oxide (>5 μm) can produce mPTP opening and cytochrome c  release.17The fact that nitric oxide could be detected by fluorescence microscopy (a relatively insensitive technique) begs the question: Was this a small or large concentration of nitric oxide, and what does it mean to downstream protection? The dilemma seems to be that if a reactive species is easily detectable, the level likely is high and potentially injurious, whereas if it is low, it may be a beneficial trigger to cytoprotective signaling but evade detection.

The current study by Jang et al.  2has focused welcomed attention on a possible role for δ-opioids in the modulation of the mPTP. Future studies will need to focus on the effects of modulators of preconditioning and postconditioning directly on the mPTP at the cellular and molecular levels. Resolution of these mito-controversies will add important information to our understanding of anesthetics as cytoprotective drugs and potential therapeutics for ischemia–reperfusion injury.

*Department of Anesthesiology, University of California, San Diego, California. †Department of Anesthesiology, University of California, San Diego, and Veterans Affairs San Diego Healthcare System, San Diego, California.

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