doi: 10.1161/01.RES.0000179773.18195.12
© 2005 American Heart Association, Inc.
Editorials |
Mitochondria and Reactive Oxygen Species
An Evolution in Function
From the Medical College of Wisconsin and VA Medical Center, Milwaukee.
Correspondence to Dr David D. Gutterman, Medical College of Wisconsin, Northwestern Mutual Professor of Cardiology, Senior Associate Dean for Research, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226. E-mail dgutterm{at}mail.mcw.edu
See related article, pages 354–362
Key Words: potassium channel • vasodilation • calcium • mitochondria • oxidative stress
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Introduction |
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 Top Introduction PreconditioningKCa Channel OpeningEndothelium-Derived...Hypoxic VasodilationeNOS ActivationReferences |
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Mitochondria are traditionally known as the energy generating centers of cells. Electron flux through the mitochondrial respiratory chain, an organized sequence of complex enzymes, hyperpolarizes the inner membrane, extruding newly generated ATP into the cytoplasm. An evolving paradigm shift has occurred in our understanding of mitochondrial function with the relatively recent observation that mitochondria are critical for the initiation of cellular apoptosis through release of cytochrome C.1 Mitochondria are also responsible for generation of substantial amounts of superoxide caused by electron leakage from the oxidative phosphorylation pathway. Reactive oxygen species (ROS) generated from mitochondria have been implicated in various forms of cell signaling in the vasculature.2
Once thought to be a toxic byproduct of cellular metabolism, ROS including superoxide and hydrogen peroxide (H2O2) also participate in a large variety of vascular cell signaling processes including activation of eNOS3 and stimulation of cell growth and migration4 through modulation of intracellular calcium,5 and activation of transcription factors such as NF-
B6 and protein kinases including ERK, p38MAPK, and Akt.7,8 Thus, physiological levels of ROS may be responsible for regulation of vascular tone9,10 and for stimulation of cell growth and migration.4
The discovery that mitochondrial function extends beyond ATP generation and that ROS may be key mediators of cellular physiology and pathology has opened new research vistas in vascular biology. Because mitochondria are responsible for the majority of ROS generated in most cells,11 linking mitochondrial respiration with ROS effects on cellular function is logical. Indeed excess release of mitochondrial oxidants has been implicated in the etiology of a host of pathologies including Alzheimer disease,12 degenerative changes in aging,13 Parkinson Disease,14 and type 2 diabetes.15
Less intense mitochondrial ROS generation has been associated with pathophysiological signaling. Mitochondrial-derived H2O2 is responsible for redox activation of c-Jun N-terminal kinase which inhibits mitochondrial metabolic enzymes.16 This serves as a potential feedback mechanism to regulate metabolic processes. In endothelial cells, H2O2 derived from mitochondria induces growth factor transactivation including receptors for vascular endothelial growth factor-2 and platelet-derived growth factor.17 These responses are inhibited by endogenous antioxidants. In human coronary arterioles, mitochondrial-derived H2O2 is responsible for flow-mediated vasodilation.18 Thus ROS are not simply a byproduct of respiration, but can serve as a control mechanism by which the mitochondria signal changes in vascular function and growth.
In the current issue of Circulation Research, Xi et al19 extend our understanding of vascular mitochondrial ROS signaling in a very important way. Previously they showed that marked depolarization of the mitochondrial membrane using the protonophore CCCP resulted in reduced calcium spark activity and thereby reduced opening of calcium-activated potassium channels (KCa) in the sarcolemma of adult rat cerebral arteriolar vascular smooth muscle cells (VSMC).20 A similar response was observed with rotenone, an inhibitor of mitochondrial complex I. Both treatments reduced the frequency of calcium sparks resulting in less KCa opening with reduced dilation. However, the link between mitochondrial membrane potential and calcium sparks was not identified.
The present study provides 2 key new findings in this regard. In contrast to the large depolarizations that reduce calcium spark activity,20 they observe an increase in calcium spark frequency and opening of KCa channels during smaller mitochondrial membrane depolarizations. Thus mitochondrial respiration can modulate vasomotor tone by dilation or constriction, establishing this organelle as a regulator of tissue perfusion. However, the cellular responses to mitochondrial membrane potential changes are complex. Large depolarizations inhibit calcium sparks, induce permeability transition pore (PTP) opening, elevate cytosolic calcium, and reduce cellular ATP. Thus responses may vary according to vascular bed and depending on the sensitivity to calcium, calcium spark frequency, and the function of the PTP. Indeed in newborn pig cerebral arteries dilation to diazoxide is not observed,21 possibly attributable to reduced calcium spark activity in neonatal cerebral vessels.22
The second major finding is that mild mitochondrial depolarization or electron transport blockade stimulates KCa through the release of ROS likely formed by mitochondrial SOD (SODII). These results identify mitochondrial ROS as a critical element in vascular signaling and implicate SODII as a potentially key regulatory enzyme in vascular function.8
The article by Xi et al19 identifies ROS as the missing link between mitochondrial depolarization and calcium spark initiation, but the specific ROS involved was not identified. H2O2 generation increased in response to diazoxide, but the critical missing experiment would have tested whether specific reduction of H2O2 alters diazoxide-induced vasodilation. Instead a manganese porphyrin SOD mimetic that also quenches H2O2 was shown to block dilation to diazoxide. By process of elimination, one may surmise that H2O2 is the responsible ROS. Superoxide has limited ability to cross membranes and is likely acted on by SOD to form H2O2. Hydroxyl radical derived from H2O2 might participate in dilation,23 but is very short-lived and reacts so promiscuously with cellular proteins that specific signaling functions are not likely.
The implications of the findings by Xi et al are broad.
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Preconditioning |
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TopIntroductionPreconditioningKCa Channel OpeningEndothelium-Derived...Hypoxic VasodilationeNOS ActivationReferences |
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This study establishes a link between the activation of 2 different classes of potassium channels. The selective mitochondrial Katp opener, diazoxide, was used to reduce mitochondrial membrane potential. Diazoxide is also a potent stimulus for myocardial ischemic preconditioning, likely operating through an NO mechanism.24 Recent evidence from Kukreja’s laboratory shows that opening of KCa also results in an early and late preconditioning.25 The article by Xi et al19 provides an alternate mechanism for diazoxide-mediated preconditioning. By lowering mitochondrial membrane potential, diazoxide releases ROS resulting in activation of KCa channels in the sarcolemma, initiating both immediate and delayed cardioprotection.
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KCa Channel Opening |
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TopIntroductionPreconditioningKCa Channel OpeningEndothelium-Derived...Hypoxic VasodilationeNOS ActivationReferences |
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Modulation of vascular KCa opening by ROS has been the subject of growing debate. In elegant studies by Toshi et al26,27 direct application of H2O2 oxidized cysteine residues on KCa, reducing channel function. Conversely, others using in vitro or in vivo preparations observe an endothelium-independent vasodilation linked to KCa channel opening.28–31 The study by Xi et al19 suggests a novel explanation for these discrepancies; namely, that H2O2 stimulates KCa opening through calcium sparks in the sarcolemma. Thus depending on the local concentration and site of formation, H2O2 could either inhibit the KCa channel directly or stimulate the potassium channel through activation of calcium sparks.
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Endothelium-Derived Hyperpolarization Factor |
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TopIntroductionPreconditioningKCa Channel OpeningEndothelium-Derived...Hypoxic VasodilationeNOS ActivationReferences |
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Recently it has been described in both mouse32 and human10,33 arterial vessels that H2O2 acts as an endothelium-derived hyperpolarization factor, mediating dilation to acetylcholine and shear stress, respectively. The dilation involves KCa activation, but global increases in intracellular calcium are not consistently seen in response to shear.34 One explanation is that H2O2 stimulates calcium sparks in underlying VSMC without a rise in cytosolic calcium. Interestingly, the H2O2 generated within human coronary endothelial cells during shear originates from mitochondrial oxidative sources.18
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Hypoxic Vasodilation |
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TopIntroductionPreconditioningKCa Channel OpeningEndothelium-Derived...Hypoxic VasodilationeNOS ActivationReferences |
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The study by Xi et al19 supports the previously forwarded idea that the mitochondria serve as a sensor for hypoxic vasodilation. Michelakis and Archer showed that H2O2 production is greater and mitochondrial respiration lower in rat pulmonary compared with renal arteries.35 ROS generation is reduced in pulmonary arteries on exposure to hypoxia, thereby decreasing outward potassium current and causing constriction. In contrast, renal arteries exposed to hypoxia generate higher levels of H2O2 and activate potassium channels to elicit dilation.
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eNOS Activation |
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TopIntroductionPreconditioningKCa Channel OpeningEndothelium-Derived...Hypoxic VasodilationeNOS ActivationReferences |
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An increase in endothelial cell calcium triggers eNOS activation and NO generation. Harrison’s group showed that H2O2 signals an increase in endothelial NO formation3,36 but the mechanism is not known. By activating calcium sparks in endothelial cells, H2O2 could raise local calcium concentrations sufficiently to activate eNOS. Supporting this speculation, vascular subsarcolemmal endoplasmic reticulum is in very close proximity to caveolae,37,38 membrane invaginations in which eNOS is concentrated. Graier et al39 elegantly showed that receptor-mediated increases in subsarcolemmal calcium occur in endothelial cells, supporting this mechanism of eNOS activation and NO generation by H2O2.
In summary, vascular mitochondria not only maintain cellular ATP but also play a key role in vascular signaling. ROS, once considered toxic byproducts of mitochondrial respiration, contribute to this important signaling function. ROS can modulate vasomotor tone, protect against ischemic damage, and promote vascular cell proliferation. This novel paradigm for vascular regulation has revealed new roles for endogenous antioxidants and modulators of mitochondrial electron transport that may be critical for vascular cell signaling.
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Acknowledgments |
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This work was supported by grants from the National Institutes of Health and a VA Merit Review. The author appreciates the expert advice of Dr Hiroto Miura.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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References |
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