New Strategies in Stroke Intervention: Ionic Transporters, Pumps, and New Channels (Contemporary Neuroscience)

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Contemporary Neuroscience

For further volumes: http://www.springer.com/series/7626

Lucio Annunziato Editor

New Strategies in Stroke Intervention Ionic Transporters, Pumps, and New Channels

Editor Lucio Annunziato Department of Neuroscience School of Medicine ‘‘Federico II’’ University of Naples Via Sergio Pansini, 5 80131 Napoli Italy [email protected]

ISBN 978-1-60761-279-7 e-ISBN 978-1-60761-280-3 DOI 10.1007/978-1-60761-280-3 Library of Congress Control Number: 2009932562 # Humana Press, a part of Springer ScienceþBusiness Media, LLC 2009 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer ScienceþBusiness Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper springer.com

Preface

Stroke is a leading cause of serious, long-term disability in the world. Every 45 seconds someone is affected by stroke and on average every 3–4 min someone dies of focal ischemia. In fact, 15 million people suffer from stroke worldwide each year. Of these, five million die and another five million are permanently disabled. Stroke ranks among the third most common cause of death after heart disease and cancer. In developed countries, the incidence of stroke is declining – largely because of the efforts to lower blood pressure and reduce smoking. However, given that age is one of the most substantiated risk factors for stroke, the aging of the world population implies a growing number of people at risk. The mean lifetime cost of a single ischemic stroke in the US is estimated at $140,048, including impatient care, rehabilitation, and follow-up care for longlasting deficits. The estimated direct and indirect costs of stroke for 2009 are $ 68.9 billion in the United States and $ 32.3 billion in the European Union countries. Stroke is caused by a blood clot that lodges in the brain and reduces oxygen supply to critical tissue. Two general therapeutic approaches are available: the first strategy is aimed at reducing the failure of arterial oxygen and glucose delivery to the local brain tissue by performing a thrombolysis of the arterial thrombus within few hours from the onset of symptoms and to reduce the tissue back-pressure occurring when failure of the blood–brain-barrier causes vasogenic edema. The second, not yet validated, therapeutical strategy is to exert a neuroprotective action on the surviving brain ischemic tissue. Despite the great effort generated in the attempt to identify new pharmacological treatments, the only current drug therapy used in the treatment of stroke belongs to the first thrombolytic strategy. However, this pharmacological approach has a narrow window of time for therapeutic application and dose-limiting adverse effects. In fact, the clot busting drug recombinant tPA can save lives and diminish disability, but its use is limited to about 3–4% of all stroke patients. In part, this is because the drug must be administered within 3 hours after the beginning of stroke symptomatology. Furthermore, tPA has also risky side effects such as haemorrhage, edema, and

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Preface

potential neurotoxic effects have been hypothesized by some experimental studies. Rupture or blockade of a blood vessel in the brain causes rapid cell death in the core of the injured region and triggers mechanisms in the surrounding area – the penumbra – that lead among several mediators to changes in the concentrations of several ions such as intracellular Ca2+, Na+, H+, K+, and radicals such as reactive oxygen species (ROS) and reactive nitrogen species (RNS). All these transductional factors might initiate cell death. In particular, it is widely accepted that a critical factor in determining neuronal death during cerebral ischemia is the progressive accumulation of intracellular Na+ ions, which can precipitate necrosis and apoptosis of vulnerable neurons. Whereas the detrimental action of [Na+]i increase is attributable to both cell swelling and microtubular disorganization – phenomena that lead to cell necrosis – a change in Ca2+,Na+, K+, H+ ions has been shown to be a key factor in ischemic brain damage, for it modulates several death pathways, including oxidative and nitrosative stress, mitochondrial dysfunction, protease activation, and apoptosis. Since Olney’s seminal work firstly suggested that excitatory amino acids could elicit neurotoxicity, a large amount of work has been accumulated showing that glutamate extracellular concentrations briskly rise during acute brain injury, thus triggering an influx of Ca2+ and Na+ ions into neurons through ionotropic glutamate receptor subtypes. This evidence has led to the elaboration of the paradigm of glutamate excitotoxicity that explains ischemic neuronal cell death as a mere consequence of Na+ and Ca2+ influx through glutamate receptors. Although this theory has been guiding basic research in the field of neurodegeneration for almost three decades, more recently it has become the object of serious criticism and reassessment. What has aroused such skepticism among researchers has been the fact that although first, second, and third generation glutamate receptor antagonists have long yielded promising results in animal models of brain ischemia, they have failed to elicit a neuroprotective action in stroke and traumatic brain injury in humans. Therefore, the theory of excitotoxicity, though a fascinating paradigm, can only explain some of the events occurring in the acute phase of anoxic insult but cannot be seen as a major target for developing new therapeutic avenues for brain ischemia. In the last decade, several seminal experimental works are markedly changing the scenario in this field. In fact, it has been shown that some integral plasma-membrane proteins, involved in the control of Ca2+, Na+, K+, H+ ions influx or efflux and, therefore, responsible for maintaining the homeostasis of these four cations, might function as crucial players in the brain ischemic process. Indeed, these proteins, by regulating Ca2+, Na+, K+, H+ homeostasis, may provide the molecular basis underlying glutamateindependent Ca2+ overload mechanisms in neuronal ischemic cell death and, most importantly, may represent more suitable molecular targets for therapeutic intervention. Targeting these mechanisms is a promising route for the development of innovative therapies for stroke treatments.

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The main goal of this book is to provide readers involved in basic and clinical research with an overview of glutamate receptor-independent channels, pumps, and ionic exchangers regulating Na+ and Ca2+ homeostasis, involved in the pathophysiology of the ischemic damage and potentially targetable in the attempt to develop a new therapy in stroke intervention. Twelve chapters are included in this book. Chapter ‘‘Basis of ionic dysregulation in cerebral ischemia’’ describes the basis of ionic dysregulation in cerebral ischemia. Chapter ‘‘Why have ionotropic and metabotropic glutamate antagonists failed in stroke therapy?’’ summarizes the reasons of the failure of the glutamate theory. Chapters ‘‘Mitochondrial channels as potential target for a pharmacological strategies in brain ischemia’’ and ‘‘Endoplasmic reticulum calcium homeostasis and neuronal pathophysiology of stroke’’ deal with the role of the two intracellular organelles, mitochondria and endoplasmic reticulum, in the pathophysiology of the ischemic event. The subsequent four chapters of the book, chapters ‘‘The Naþ/Ca2þ exchanger: a target for therapeutic intervention in cerebral ischemia’’, ‘‘The ‘loop’ diuretic drug bumetanide-sensitive Naþ-Kþ-Cl– cotransporter in cerebral ischemia’’, ‘‘The Naþ/Hþ exchanger: A target for therapeutic intervention in cerebral ischemia’’, and ‘‘The Naþ/Kþ-ATPase as a drug target for ischemic stroke’’, present data regarding the potential involvement of ionic transporters responsible for the control of neuronal ionic homeostasis in the development of stroke lesion. In particular, the following transporters are described: Naþ/ Ca2þ exchangers, NCXs, Na+/K+/Ca2+/Cl– cotransporters, NKCCs, Na+/ H+ Exchangers, NHEs, and Naþ/Kþ ATPase. In the second part of the book, chapters ‘‘Acid-sensing ion channels (ASICs): New targets in stroke treatment’’, ‘‘Role of TRPM7 in ischemic CNS injury’’, ‘‘Subtypes of voltage-gated Ca2+ channels and ischemic brain injury’’, and ‘‘The diverse roles of K+ channels in brain ischemia’’, it is described the role of important membrane ionic channels such as Acid-Sensing Ionic Channels, ASIC, the Transient Receptor Potential Channels, TRPC; Voltage Operated Ca2+ Channels, VOCC and K+ channels. Each chapter dedicated to most of the molecular targets involved in the maintenance of ionic homeostasis describes in a coordinated way the relevant information related to: (1) gene structure, (2) structural features, (3) molecular biology, (4) cellular and tissue distribution, (5) biophysical and electrophysiological properties, (6) receptorial, transcriptional and transductional regulatory mechanisms, (7) physiological properties, (8) pathophysiological relevance in stroke, (9) pharmacological modulation, (10) preliminary clinical trials, and (11) therapeutic perspectives. This book written by the most authoritative scientists in the field should provide to the readers the most relevant information to understand the role played by the most recently discovered mechanisms involved in the pathophysiology of stroke thus providing the characterization of new pharmacological avenues for the cure of this relevant neurological disease. Naples, Italy

Lucio Annunziato

Acknowledgments

We thank Dr. Paola Merolla for editorial revision, Ms Giuliana Pellegrini of the Art Studio Design ‘‘Ciotola’’ for revising all the figures of the book, and Dr. Fabrizio Esposito, Associate Researcher at the Department of Neuroscience, School of Medicine, ‘‘Federico II’’ University of Naples, for providing us with the fMRI image of human brain used in the cover of the present book.

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Contents

Basis of Ionic Dysregulation in Cerebral Ischemia. . . . . . . . . . . . . . . . . . . Thiruma V. Arumugam, Eitan Okun, and Mark P. Mattson

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Why have Ionotropic and Metabotropic Glutamate Antagonists Failed in Stroke Therapy? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gianfranco Di Renzo, Giuseppe Pignataro, and Lucio Annunziato

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Mitochondrial Channels as Potential Targets for Pharmacological Strategies in Brain Ischemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rosemary H. Milton and Michael R. Duchen

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Endoplasmic Reticulum Calcium Homeostasis and Neuronal Pathophysiology of Stroke. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alexei Verkhratsky

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The Naþ/Ca2þ Exchanger: A Target for Therapeutic Intervention in Cerebral Ischemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lucio Annunziato, Pasquale Molinaro, Agnese Secondo, Anna Pannaccione, Antonella Scorziello, Giuseppe Pignataro, Ornella Cuomo, Rossana Sirabella, Francesca Boscia, Alessandra Spinali, and Gianfranco Di Renzo The ‘‘Loop’’ Diuretic Drug Bumetanide-Sensitive Naþ-Kþ-Cl– Cotransporter in Cerebral Ischemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dandan Sun

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The Naþ/Hþ Exchanger: A Target for Therapeutic Intervention in Cerebral Ischemia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 Jin Xue and Gabriel G. Haddad The Naþ/Kþ-ATPase as a Drug Target for Ischemic Stroke. . . . . . . . . . . Melissa A. Gottron and Donald C. Lo

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Contents

Acid-Sensing Ion Channels (ASICs): New Targets in Stroke Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Giuseppe Pignataro Role of TRPM7 in Ischemic CNS Injury . . . . . . . . . . . . . . . . . . . . . . . . . . Michael F. Jackson, Hong-Shuo Sun, Michael Tymianski and John F. MacDonald

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Subtypes of Voltage-Gated Ca2þ Channels and Ischemic Brain Injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Soon-Tae Lee, Daejong Jeon, and Kon Chu

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The Diverse Roles of Kþ Channels in Brain Ischemia . . . . . . . . . . . . . . . . Hiroaki Misonou and James S. Trimmer

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Clinical Trials with Drugs Targeting Ionic Channels, Antiporters, and Pumps in Ischemic Stroke . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alessandra Spinali, Giuseppe Pignataro, Gianfranco Di Renzo, and Lucio Annunziato Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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List of Contributors

Lucio Annunziato Division Pharmacology, Department of Neuroscience, School of Medicine, ‘‘Federico II’’ University of Naples, Italy, [email protected] Thiruma V. Arumugam Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX 79106, USA, [email protected] Francesca Boscia Division Pharmacology, Department of Neuroscience, School of Medicine, ‘‘Federico II’’ University of Naples, Italy, [email protected] Kon Chu Stroke & Stem Cell Laboratory, Department of Neurology, Clinical Research Institute, Comprehensive Epilepsy Center, Seoul National University Hospital, Program in Neuroscience, Seoul National University, Seoul, 110-744, South Korea, [email protected] Ornella Cuomo Division Pharmacology, Department of Neuroscience, School of Medicine, ‘‘Federico II’’ University of Naples, Italy, [email protected] Gianfranco Di Renzo Division Pharmacology, Department of Neuroscience, School of Medicine, ‘‘Federico II’’ University of Naples, Italy, [email protected] Michael R. Duchen Department of Physiology, University College London, London WC1E 6BT, UK, m.duchen @ucl.ac.uk Melissa A. Gottron Center for Drug Discovery and Department of Neurobiology, Duke University Medical Center, Durham, NC 27710, USA, [email protected] Gabriel G. Haddad Departments of Pediatrics and Neuroscience, University of California San Diego, La Jolla, CA 92093, USA, and Rady Children’s Hospital, San Diego, CA 92123, USA, [email protected] Michael F. Jackson Robarts Research Institute, Molecular Brain Research Group, University of Western Ontario, London, ON N6A 5K8, Canada, [email protected] xiii

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Daejong Jeon Stroke & Stem Cell Laboratory, Department of Neurology, Clinical Research Institute, Comprehensive Epilepsy Center, Seoul National University Hospital, Program in Neuroscience, Seoul National University, Seoul, 110-744, South Korea, [email protected] Soon-Tae Lee Stroke & Stem Cell Laboratory, Department of Neurology, Clinical Research Institute, Comprehensive Epilepsy Center, Seoul National University Hospital, Program in Neuroscience, Seoul National University, Seoul, 110-744, South Korea, [email protected] Donald C. Lo Center for Drug Discovery and Department of Neurobiology, Duke University Medical Center, Durham, NC 27710, USA, [email protected] John F. MacDonald Robarts Research Institute, Molecular Brain Research Group, University of Western Ontario, London, ON N6A 5K8, Canada, and Department of Physiology, University of Toronto, King’s College Circle, Toronto, ON M5S 1A8, Canada, [email protected] Mark P. Mattson Laboratory of Neurosciences, National Institute on Aging Intramural Research Program, Baltimore, MD 21224, USA, [email protected] Rosemary H. Milton Department of Physiology, University College London, Gower Street, London WC1E 6BT, UK, [email protected] Hiroaki Misonou Department of Neural and Pain Sciences, Program in Neuroscience, Dental School, University of Maryland, Baltimore, MD 21201, USA, [email protected] Pasquale Molinaro Division Pharmacology, Department of Neuroscience, School of Medicine, ‘‘Federico II’’ University of Naples, Italy, [email protected] Eitan Okun Laboratory of Neurosciences, National Institute on Aging Intramural Research Program, Baltimore, MD 21224, USA, [email protected] Anna Pannaccione Division Pharmacology, Department of Neuroscience, School of Medicine, ‘‘Federico II’’ University of Naples, Italy, [email protected] Giuseppe Pignataro Division Pharmacology, Department of Neuroscience, School of Medicine, ‘‘Federico II’’ University of Naples, Italy, [email protected] Antonella Scorziello Division Pharmacology, Department of Neuroscience, School of Medicine, ‘‘Federico II’’ University of Naples, Italy, [email protected]


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Agnese Secondo Division Pharmacology, Department of Neuroscience, School of Medicine, ‘‘Federico II’’ University of Naples, Italy, [email protected] Alessandra Spinali Division Pharmacology, Department of Neuroscience, School of Medicine, ‘‘Federico II’’ University of Naples, Italy, [email protected] Rossana Sirabella Division Pharmacology, Department of Neuroscience, School of Medicine, ‘‘Federico II’’ University of Naples, Italy, [email protected] Dandan Sun Department of Neurological Surgery, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705, USA, [email protected] Hong-Shuo Sun Department of Physiology, University of Toronto, King’s College Circle, Toronto, ON M5S 1A8, Canada, and Toronto Western Hospital Research Institute, Toronto, ON M5T 2S8, Canada, [email protected] James S. Trimmer Department of Neurobiology, Physiology, and Behavior, College of Biological Sciences; and Department of Physiology and Membrane Biology, School of Medicine, University of California, Davis, CA 95616, USA [email protected] Michael Tymianski Department of Physiology, University of Toronto, King’s College Circle, Toronto, ON M5S 1A8, Canada, and Toronto Western Hospital Research Institute, Toronto, ON M5T 2S8, Canada, [email protected] Alexei Verkhratsky Faculty of Life Sciences, The University of Manchester, Manchester M13 9PT, UK and Institute of Experimental Medicine, ASCR, 142 20 Prague 4, Czech Republic, [email protected] Jin Xue Departments of Pediatrics, University of California San Diego, La Jolla, CA 92093, US, [email protected]

Basis of Ionic Dysregulation in Cerebral Ischemia Thiruma V. Arumugam, Eitan Okun, and Mark P. Mattson

1 Introduction Stroke causes 9% of all deaths around the world and is the second most common cause of death after ischemic heart disease. Enhancing recovery from stroke and limiting ischemic damage are major goals to decrease stroke morbidity and mortality [15]. Brain tissue is extremely sensitive to oxygen and glucose deprivation, and even brief ischemia can initiate a complex sequence of events that ultimately culminates in cellular death. Studies performed during the past 30 years have identified several key pathophysiological events that lead to ischemic neuronal degeneration. The pathophysiological processes in stroke are complex and involve disruption of the blood–brain barrier (BBB), energy failure, loss of cell ion homeostasis, acidosis, increased intracellular calcium levels, excitotoxicity, free radical-mediated toxicity, generation of arachidonic acid products, cytokine-mediated apoptosis, activation of glial cells, and infiltration of leukocytes [39, 5, 6]. In focal cerebral ischemia, ischemic tissue is divided into an infarction core and penumbra. The core is the area of the brain where blood flow is reduced below 10–20% of its normal levels [55]. In the core, rapid anoxic depolarization causes immediate loss of membrane potential followed by the loss of membrane integrity and rapid necrotic cell death. The penumbra is the tissue surrounding the core where the blood flow is partly preserved due to collateral circulation and diffusion [55]. The decline in blood flow and the accompanying loss of oxygen supply result in a reduction of high-energy metabolites such as adenosine triphosphate (ATP) and phosphocreatine [39]. The combination of ATP breakdown and compensatory activation of anaerobic glycolysis during ischemia leads to an increase in the levels of inorganic phosphate, lactate, and Hþ formation causing cellular acidification. The decrease of cellular ATP levels impairs the ability of T.V. Arumugam (*) School of Biomedical Sciences, The University of Queensland, Brisbane, QLD 4072, Australia e-mail: [email protected]

L. Annunziato (ed.), New Strategies in Stroke Intervention, Contemporary Neuroscience, DOI 10.1007/978-1-60761-280-3_1, Ó Humana Press, a part of Springer ScienceþBusiness Media, LLC 2009

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membrane ion-motive ATPases to remove Naþ and Ca2þ from the cell [2]. This results in membrane depolarization, which promotes activation of synaptic glutamate receptors (Fig. 1). Neurons are normally exposed to brief pulses of glutamate because excess extracellular glutamate is actively returned to presynaptic terminals and glial cells. During ischemia, however, the energydependent mechanisms responsible for glutamate re-uptake are impaired, hence extracellular glutamate levels can reach 100 mM. Excessive accumulation of extracellular glutamate further activates glutamate receptors, resulting in massive Ca2þ influx through N-methyl-D-aspartate (NMDA) receptors, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, and voltage-dependent Ca2þ channels [43, 24]. This elevation of extracellular glutamate causes a prolonged and excessive activation of membrane glutamate receptors, further stimulating Ca2þ influx. Mitochondrial dysfunction occurs as the result of energy failure and disruption of cellular Ca2þ homeostasis.

Fig. 1 Ionic dysregulation during cerebral ischemia. The decrease of cellular ATP levels impairs the ability of membrane ion-motive ATPases to remove Naþ and Ca2þ from the cell. The increase in cytosolic Naþ causes cell swelling. Increased levels of intracellular Naþ can directly cause oxidative stress and cell death. When intracellular Naþ, Cl–, and H2O increase occur at the same time, cytotoxic edema occurs. Excessive Kþ efflux and intracellular Kþ depletion play a role in the apoptotic cascade. Kþ release from mitochondria contributes to the increased ROS production and ATP depletion. Ca2þ influx induces activation of two distinct cysteine proteases: caspases and calpains. These in turn cause degradation of cytoskeletal proteins, membrane receptors, and metabolic enzymes. Ca2þ also induces oxidative stress, which along with caspases induces mitochondrial dysfunction that results in cell death by apoptosis

Basis of Ionic Dysregulation in Cerebral Ischemia

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Increased production of free radicals results from mitochondrial dysfunction, Ca2þ overload, and activation of enzymes such as cyclooxygenase and nitric oxide synthase [39]. Free radicals damage cellular proteins, DNA, and membrane lipids. A particularly important aspect of oxidative stress in neurons is membrane lipid peroxidation, which results in the generation of toxic aldehydes such as 4-hydroxynonenal that impair the function of membrane ion-motive ATPases and glucose and glutamate transporters, thereby amplifying disruption of cellular calcium homeostasis [36]. The traditional view that glutamate-mediated disruption of cellular Ca2þ homeostasis in neurons has been expanded by a number of observations that implicate Cl channels and several types of non-channel transporter proteins, such as the Naþ/Ca2þ exchangers (NCXs), Kþ-dependent Naþ/Ca2þ exchangers (NCKXs), Naþ/Kþ/Ca2þ/Cl cotransporters (NKCCs), and Naþ/Hþ exchangers (NHEs) in the development of stroke lesion [2, 21, 11, 14]. Some of these ion transporters increase tissue damage by promoting pathological cell swelling and necrotic cell death, while others contribute to a long-term accumulation of cytoplasmic Ca2þ. Apart from the above-mentioned ion transporters, recently identified channels such as acid-sensing ionic channels (ASICs) [58], the sulfonylurea receptors (SUR1s) [52], TWIK-related potassium channels (TREKs) [8], and the transient receptor potential channels (TRPCs) [30] are also implicated in stroke pathology. This chapter summarizes the mechanisms of disruption of Ca2þ, Naþ, Kþ, and Cl homeostasis in cerebral ischemia.

2 Disruption of Ca2þ Homeostasis in Cerebral Ischemia Studies of stroke patients and animal and cell culture models have provided a wealth of data supporting the involvement of alterations in Ca2þ regulation in the pathogenesis of stroke. Activation of glutamate receptors and voltagedependent Ca2þ channels (VDCCs) and impairment of NCXs and ion-motive ATPases contribute to neuronal Ca2þ overload and cell death after a stroke [22]. Therefore, stabilization of intracellular Ca2þ has been a major goal in the search for a therapeutic method of minimizing the brain injury that follows stroke. Neurons are excitable cells that rapidly transfer electrochemical signals in a highly controlled spatio-temporal manner. A major intracellular messenger that mediates many physiological responses of neurons is Ca2þ. When properly controlled, Ca2þ fluxes across the plasma membrane and between intracellular compartments play critical roles in fundamental functions of neurons, including the regulation of neurite outgrowth and synaptogensis, synaptic transmission and plasticity, and cell survival [34]. The influx of Ca2þ through voltagedependent and ligand-gated channels in the plasma membrane is a critical signal for the release of neurotransmitters from presynaptic terminals and for responses of the postsynaptic neuron [37]. Glutamate, the major excitatory neurotransmitter in the central nervous system (CNS), induces an increase in

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the concentration of cytoplasmic Ca2þ by directly activating AMPA and NMDA receptor channels and by indirectly activating VDCC [34, 48]. In addition, activation of metabotropic glutamate receptors coupled to the GTPbinding protein Gq11 stimulates the release of inositol triphosphate (IP3), which activates Ca2þ channels in the endoplasmic reticulum (ER) [17]. Ca2þ is removed from the cytoplasm by the activities of the plasma membrane NCXs, plasma membrane and ER Ca2þ-ATPases, and Ca2þ-binding proteins such as calbindin and parvalbumin [38, 40]. By buffering intracellular Ca2þ loads, Ca2þ-binding proteins such as calbindin may serve as endogenous antiexcitotoxic proteins [40]. Ca2þ can also be transported into and released from mitochondria [56]. During normal physiological activity, the intracellular Ca2þ concentration increases only transiently (seconds to a few minutes) and has no adverse effects on the neurons [53]. However, in cerebral ischemia the ability of neurons to control Ca2þ fluxes and recover from a Ca2þ load is compromised. Excessive Ca2þ levels induce neuronal cell death in several different crossamplifying cascades: (1) Ca2þ activates either directly or indirectly cysteine proteases called calpains and caspases that degrade a variety of substrates, including cytoskeletal proteins, membrane receptors, and metabolic enzymes [10]. Calpains may also play an important role in the triggering of apoptotic cascades by virtue of their ability to activate caspases [28]. (2) Ca2þ induces oxidative stress [35] through several different mechanisms. This includes activation of oxygenases, perturbation of mitochondrial Ca2þ and energy metabolism, and induction of membrane-associated oxidative stress (MAOS). The reactive oxygen species (ROS) generated in response to glutamate-induced Ca2þ influx includes superoxide anion radicals, hydrogen peroxide, hydroxyl radicals, nitric oxide, and peroxynitrite [32]. (3) Ca2þ triggers apoptosis, a form of programmed cell death [1]. This might occur by Ca2þ -mediated induction/activation of pro-apoptotic proteins such as Bax, Par-4, and p53 leading to mitochondrial membrane permeability changes, release of cytochrome c, and caspase activation [13, 12].

3 Disruption of Naþ Homeostasis in Cerebral Ischemia Maintenance of a low intracellular Naþ is critical for normal cell function. Under steady-state conditions, intracellular Naþ is generally maintained below 20 mM whereas the extracellular Naþ concentration is 140 mM. This sevenfold concentration difference, together with the negative membrane potential (Vm), constitutes a substantial inward driving force for Naþ, which is used ubiquitously to drive a wide variety of transport processes. Cell volume and net intracellular osmolyte concentration play a pivotal role in a wide range of cellular processes and consequently must be tightly regulated to maintain normal cell function. In healthy cells, intracellular Naþ is maintained relatively


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constant due to Naþ-Kþ-ATPase activity [4, 42]. Although excitotoxicity is frequently considered synonymous with Ca2þ-dependent cell death, early studies clearly showed neurotoxic effects of glutamate such as rapid toxicity that are determined mainly by the influx of Naþ, Cl–, as well as osmotic swelling, and a more delayed Ca2þ-dependent cell death [57, 23]. One form of excitotoxicity relies on the presence of extracellular Naþ and Cl but not Ca2þ, and manifests as rapidly forming dendritic varicosities followed by generalized somatic swelling and necrotic cell death. Replacement of extracellular Naþ or Cl with impermeant ion species prevents glutamate-induced cell swelling and strongly reduces cell death. Pharmacological evidence suggests that Naþ enters the cell mainly via the glutamate-gated AMPA and NMDA channels [42, 20]. Increases in intracellular Naþ during cerebral ischemia favor NCX-mediated Ca2þ influx and consequent Ca2þ-induced cell damage [29]. The rapid increases in intracellular Naþ and Ca2þ together with swelling of neurons, astrocytes, and endothelial cells are major causes of brain damage associated with limited perfusion. Uncontrolled cell swelling is often associated with necrosis and is harmful to neuronal and glial cells [44]. The extent of intracellular Naþ elevation and cell swelling varies depending on the location in the ischemic region, with the most pronounced effects occurring in the core of the ischemic zone and more moderate slower onset effects in the penumbral regions. During cerebral ischemia, brain edema forms by a process involving increased secretion of Naþ and water across an intact brain blood barrier (BBB) [49]. At the same time, ischemia stimulates NHE1and NKCC1-dependent uptake of Naþ Cl, and water, causing cytotoxic edema [29, 47, 33], a process likely facilitated by the increased BBB secretion of Naþ, Cl, and water into the brain interstitium. Apart from Naþ influx and accumulation in the intracellular space-inducing cell swelling, increased levels of intracellular Naþ can directly cause damage, oxidative stress, and cell death. When oxidative stress occurs together with a Naþ influx, the damaging effect of oxidative stress is greatly exacerbated. The basis for this is an increased utilization of ATP by the Naþ pump activated by a Naþ entry coupled with the inability of mitochondria to respond adequately with increasing ATP production. An increase in intracellular Naþ through voltage-sensitive sodium channels (VSSCs) during hypoxia contributes to apoptosis [7]. Hypoxia increases intracellular Naþ and induces neuronal apoptosis. Reducing Naþ influx with the VSSC blocker, tetrodotoxin (TTX), attenuates apoptotic neuronal death via a reduction in caspase-3 activation [7]. Activation of VSSCs during hypoxia causes altered expression of genes that are critical regulators of apoptotic cell death [7].

4 Disruption of Kþ Homeostasis in Cerebral Ischemia Extracellular Kþ can range within 2.5–3.5 mM under normal conditions to 50–80 mM under ischemic and spreading depression events. Sustained exposure to elevated Kþ has been shown to cause significant neuronal death even under

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conditions of abundant glucose supply [54]. Physiological concentrations of potassium prevent death receptor activation, cytochrome c release, apoptosome formation, caspase activation, and apoptotic nuclease activity [59]. Kþ channels are a major contributor to a cell’s resting potential and their activation helps to maintain a hyperpolarized resting membrane potential. Kþ efflux occurs prior to Naþ or Ca2þ influx [27], which does not occur until ATP levels have fallen by more than 50%. There are a number of different types of Kþ channels. The metabolic nature of an ischemic insult suggests that ATP-sensitive Kþ channels, which are activated by a decrease in ATP, would be one of the first channels to respond during ischemia [60]. Astrocytes attempt to buffer this increase in Kþ efflux by switching to anaerobic glycolysis and swell 5–10 times their normal size. Eventually, astrocytes are no longer able to cope with the increase in Kþ efflux and they lyse. It was shown that cortical neurons exposed to glutamate with reduced Naþ and Ca2þ substantially lose their intracellular Kþ and undergo apoptosis. Both Kþ loss and apoptosis can be attenuated by increasing extracellular Kþ, indicating that glutamate receptor-mediated Kþ efflux contributes to neuronal apoptosis after brain ischemia [60]. Excessive Kþ efflux and intracellular Kþ depletion have been hypothesized to be key steps in the apoptotic cascade of many cells, including central neurons. One possible factor limiting the benefit of enhancing Kþ channel activity in the ischemic brain might be enhancement of an apoptotic component of focal ischemic neuronal death. Potassium channel openers are neuroprotective against ischemic cell death in rodents, presumably as a result of membrane hyperpolarization and reduced membrane excitability [59]. Kþ channel openers may interfere with Ca2þ mobilization from intracellular stores; in addition, these openers may mimic preconditioning or alter KATP channels on mitochondrial membranes [59]. Kþ release from mitochondria, and subsequent efflux from the cell, occurs in neurons subjected to ischemic insults that induce apoptosis. The ability of 5-hydroxydecanoate (5HD), an inhibitor of mitochondrial Kþ channels, to reduce ischemic brain injury in vivo and apoptosis of cultured neurons suggests a role for modulation of mitochondrial KATP (Mito-KATP) in neuronal death cascades. The protective effect of 5HD against focal ischemic brain injury was only observed when 5HD was administered prior to the onset of ischemia, suggesting that opening of Mito-KATP soon after the onset of ischemia plays an important role in the cell death cascade. During ischemia, Mito-KATP channels may be open and contribute to the increased ROS production and ATP depletion [31].

5 Disruption of Cl Homeostasis in Cerebral Ischemia Anion channels play an electrogenic role in excitable cells, shifting the membrane potential. Recent studies have demonstrated more general roles for anion channels. These include roles in cell volume regulation, cell proliferation, and cell death


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[26, 25]. In adult mammalian neurons, Clconcentration difference between the extracellular milieu (110–150 mM) and the intracellular space (7–30 mM) generates a gradient that is maintained by several Cl extrusion mechanisms, including Kþ–Cl cotransporters, Naþ-dependent Cl/HCO3– exchangers, ClATPase, and VDCCs [51]. Following cerebral ischemia, intracellular Cl increases by several mechanisms, including passive influx to accompany the influx of Naþ, influx through GABA-gated Cl– channels, inhibition of the voltage-gated Cl channel, hypofunction or reversal of outward Clcotransporters, and activation of inward Clcotransporters [45, 51, 11]. In in vitro models of ischemia, an increase in intracellular Cl occurs in area CA1 cell as well as CA1 pyramidal neurons and interneurons early following the onset of re-oxygenation [50]. Similar increases in intracellular Cl have been observed in hypoglossal neurons subjected to anoxia [19]. GABA, an inhibitory neurotransmitter, is usually hyperpolarizing under cerebral ischemia, because the equilibrium reversal potential for Cl (ECl) is negative relative to the resting membrane potential. In some instances, intracellular Cl– levels are higher than normal and ECl shifts to more positive values. If ECl is more positive than the resting membrane potential, GABAA receptor activation causes Cl efflux and membrane depolarization [51]. The increase in intracellular Cl following energy deprivation is detrimental to the neurons. For example, reduced GABAA responses in area CA1 pyramidal neurons are observed following the oxygen–glucose deprivation–induced rise in intracellular Cl [50, 18]. These in vitro findings are supported by experiments with animal models of cerebral ischemia. In rats subjected to focal cerebral ischemia, there is a depolarizing shift in the reversal potential for GABAA-mediated inhibitory postsynaptic potentials [41]. When ATP levels are low during cerebral ischemia, Cl–ATPase fails to transport Cl into the extracellular space. This may result in ATPdependent rundown of GABAA currents in neurons [16]. Increase in extracellular Kþ levels during ischemia may drive both Kþ and Cl back into the neuron [46]. Elevations in extracellular Kþ have been shown to reduce the GABA-mediated inhibitory postsynaptic potential due to a positive shift in the ECl [9] and to stimulate the Naþ/Kþ/Cl cotransporter to increase intracellular Cl [51].

6 Conclusion Cell culture and animal models of ischemic brain injury have proven invaluable in elucidating the cellular and molecular mechanisms of neuronal cell death in stroke. The failure of ionic homeostasis is a hallmark of ischemic brain damage. Cell surface signaling events include the activities of voltage-gated Kþ, Naþ, and Ca2þ channels and ligand-gated glutamate and GABA receptors and channels. Ca2þ channels have received a lot of attention in studies of cerebral ischemia because Ca2þ influx and the disruption of Ca2þ homeostasis play an important role in ischemic cell death. Intracellular signaling events include

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alterations in cytosolic and subcellular Ca2þ dynamics, Ca2þ-dependent kinases, and immediate early genes whereas intercellular mechanisms include mitochondrial dysfunction and free radical formation. Kþ channels are major contributors to a cell’s resting potential and their activation helps to maintain a hyperpolarized resting membrane potential. Naþ channels also play an important role in neuronal excitability and they are as widely expressed as Kþ channels. Failure of both Kþ and Naþ channels in cerebral ischemia causes significant neuronal death. In addition intracellular Cl increases by number of different mechanisms. Pathological accumulation of Naþ and Cl plays a very important role in the development of ischemic cell and tissue injury. The damaging effects of high cytosolic Naþ and Cl are realized via pathological cell swelling, the impact of Naþ overload on Ca2þ homeostasis, and an increased metabolic burden that is placed on the cell due to enhanced work of the Naþ, Kþ, and Ca2þ pumps. Effective stroke therapies may indeed require targeting several points in the neurotoxic cascade in a specific temporal sequence. Such a combination therapy may begin with the immediate administration of clot-dissolving tPA, followed by a Ca2þ channel blocker, as well as inhibitors blocking Naþ, Kþ, and Cl transport pathways.

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32. Love S. Oxidative stress in brain ischemia. Brain Pathol 9: 119–131, 1999. 33. Luo J, Kintner DB, Shull GE, and Sun D. ERK1/2-p90RSK-mediated phosphoryl tion of Na+/H+ exchanger isoform 1. A role in ischemic neuronal death. J Biol Chem 282: 28274–28284, 2007. 34. Mattson MP. Neurotransmitters in the regulation of neuronal cytoarchitecture. Brain Res 472: 179–212, 1988. 35. Mattson MP. Modification of ion homeostasis by lipid peroxidation: roles in neuronal degeneration and adaptive plasticity. Trends Neurosci 21: 53–57, 1998. 36. Mattson MP. Excitotoxic and excitoprotective mechanisms: abundant targets for the prevention and treatment of neurodegenerative disorders. Neuromolecular Med 3: 65–94, 2003. 37. Mattson MP. Calcium and neurodegeneration. Aging Cell 6: 337–350, 2007. 38. Mattson MP, Guthrie PB, and Kater SB. A role for Na+-dependent Ca2+ extrusion in protection against neuronal excitotoxicity. FASEB J 3: 2519–2526, 1989. 39. Mattson MP, Culmsee C, and Yu ZF. Apoptotic and antiapoptotic mechanisms in stroke. Cell Tissue Res 301: 173–187, 2000. 40. Mattson MP, Rychlik B, Chu C, and Christakos S. Evidence for calcium-reducing and excito-protective roles for the calcium-binding protein calbindin-D28k in cultured hippocampal neurons. Neuron 6: 41–51, 1991. 41. Mittmann T, Qu¨ M, Zilles K, and Luhmann HJ. Long-term cellular dysfunction after focal cerebral ischemia: in vitro analyses. Neuroscience 85: 15–27, 1998. 42. Mongin AA. Disruption of ionic and cell volume homeostasis in cerebral ischemia: The perfect storm. Pathophysiology 14: 183–193, 2007. 43. Nakamichi N, Oikawa H, Kambe Y, and Yoneda Y. Relevant modulation by ferrous ions of N-methyl-D-aspartate receptors in ischemic brain injuries. Curr Neurovasc Res 1: 429–440, 2004. 44. Okada Y, Maeno E, Shimizu T, Dezaki K, Wang J, and Morishima S. Receptor-mediated control of regulatory volume decrease (RVD) and apoptotic volume decrease (AVD). J Physiol 532: 3–16, 2001. 45. Okada Y, Shimizu T, Maeno E, Tanabe S, Wang X, and Takahashi N. Volume-sensitive chloride channels involved in apoptotic volume decrease and cell death. J Membr Biol 209: 21–29, 2006. 46. Payne JA. Functional characterization of the neuronal-specific K-Cl cotransporter: implications for [K+]o regulation. Am J Physiol 273: C1516–C1525, 1997. 47. Pedersen SF, O’Donnell ME, Anderson SE, and Cala PM. Physiology and pathophysiology of Na+/H+ exchange and Na+-K+-2Cl– cotransport in the heart, brain, and blood. Am J Physiol Regul Integr Comp Physiol 291: R1–R25, 2006. 48. Pinheiro PS and Mulle C. Presynaptic glutamate receptors: physiological functions and mechanisms of action. Nat Rev Neurosci 9: 423–436, 2008. 49. Rosenberg GA. Ischemic brain edema. Prog Cardiovasc Dis 42: 209–216, 1999. 50. Sah R and Schwartz-Bloom RD. Optical imaging reveals elevated intracellular chloride in hippocampal pyramidal neurons after oxidative stress. J Neurosci 19: 9209–9217, 1999. 51. Schwartz-Bloom RD and Sah R. Gamma-aminobutyric acid(A) neurotransmission and cerebral ischemia. J Neurochem 77: 353–371, 2001. 52. Simard JM, Tarasov KV, and Gerzanich V. Non-selective cation channels, transient receptor potential channels and ischemic stroke. Biochim Biophys Acta 1772: 947–957, 2007. 53. Takahashi A, Camacho P, Lechleiter JD, and Herman B. Measurement of intracellular calcium. Physiol Rev 79: 1089–1125, 1999. 54. Wei L, Yu SP, Gottron F, Snider BJ, Zipfel GJ, and Choi DW. Potassium channel blockers attenuate hypoxia- and ischemia-induced neuronal death in vitro and in vivo. Stroke 34: 1281–1286, 2003. 55. Weinstein PR, Hong S, and Sharp FR. Molecular identification of the ischemic penumbra. Stroke 35: 2666–2670, 2004.


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Why have Ionotropic and Metabotropic Glutamate Antagonists Failed in Stroke Therapy? Gianfranco Di Renzo, Giuseppe Pignataro, and Lucio Annunziato

1 Introduction The concept of ‘‘excitotoxicity’’ was introduced in 1969 when Olney and Sharpe first demonstrated that neurons exposed to their own neurotransmitter glutamate were destined to die [49]. Later on, in 1985, glutamate toxicity was associated with anoxic cell death, since anoxic depolarization resulted in the release of glutamate into the extracellular compartments [42, 54]. Similarly, in 1987, Choi indicated that glutamate was a remarkably potent and rapidly acting neurotoxin able to mediate neurotoxic effects by inducing Ca2þ influx through glutamate receptor activation, and he supported the theory that glutamate can be considered a key neurotransmitter in developing many neurological diseases [13, 14, 15]. Since then, glutamate receptors have been the most studied channels involved in ischemic stroke pathophysiology. Glutamate can exert its effects by interacting with both ionotropic glutamate receptors (iGluRs), also referred to as ligand-gated ion channels, and metabotropic glutamate G-protein-coupled receptors. The group of iGluRs comprises three major classes, the a-amino-3hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, kainate (KA) receptors, and N-methyl-D-aspartate (NMDA) receptors, named according to their selective agonists [62]. Glutamatergic synapses frequently harbor both AMPA and NMDA receptors. Characteristically, both classes of receptors differ in their response kinetics upon presynaptic glutamate release. Indeed, AMPA receptors mediate fast glutamate-gated postsynaptic responses, even at very negative potentials or in the absence of action potentials. The fast desensitization of AMPA receptors leads to short excitatory postsynaptic currents (EPSCs). In contrast, NMDA receptors contain an agonist-binding site, i.e., a glycine modulatory site, and other binding sites within the ion channel, where magnesium exerts a voltage-dependent block [21]. Acting as detectors of G. Di Renzo (*) Division Pharmacology, Department of Neuroscience, School of Medicine, ‘‘Federico II’’ University of Naples, Via Pansini 5, 80131, Naples, Italy e-mail: [email protected]


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membrane depolarization and ligand-gated channel activation, NMDA receptors require the removal of the Mg2þ block by an increase in membrane potential to allow cation permeation through the receptor pore [62]. The excitotoxicity theory encountered a great favor for almost 40 years, because of a discrete success of glutamate receptors antagonists in treating experimentally induced stroke. Unfortunately, however, over the last two decades, all stroke clinical trials with agents acting on these receptors have been disappointing [16, 17, 45, 46, 48]. The disappointing experience derived from the use of pharmacological agents interfering with glutamate-mediated mechanisms in stroke pathophysiology has been paid by many scientists working in the field with the reluctance of government and private associations to grant projects on the development of new drugs to treat stroke. Thus, the discouraging results have redirected scientists’ attention to other channels and membrane transporters able to control neuronal ionic homeostasis. The purpose of this chapter is to review some of the evidence in the field in an attempt to explain why drugs acting on glutamate receptors have resulted ineffective in treating stroke.

2 Glutamate and Stroke It is widely accepted that a critical factor in determining neuronal and glial death during cerebral ischemia is the progressive accumulation of intracellular Naþ([Naþ]i) and Ca2þ([Ca2þ]i) ions, which can precipitate necrosis and apoptosis of vulnerable neurons. Whereas the detrimental action of [Naþ]i increase is attributable to both cell swelling and microtubular disorganization – two phenomena that lead to cell necrosis [59] – a rise in [Ca2þ]i has been shown to be a key factor in ischemic brain damage, for it modulates several death pathways, including oxidative and nitrosative stress, mitochondrial dysfunction, and protease activation. Since Olney’s seminal work first suggested that excitatory amino acids could elicit neurotoxicity [49], a vast amount of data have demonstrated that glutamate extracellular concentrations briskly rise during acute brain injury, thus triggering an influx of Ca2þ and Naþ ions into neurons through glutamate receptors [13]. This evidence has led to the elaboration of the paradigm of glutamate excitotoxicity, a theory that explained ischemic neuronal cell death as a mere consequence of Naþ and Ca2þ influx through glutamate receptors [13]. Although this paradigm has been guiding basic research in the field of neurodegeneration for almost three decades, more recently it has become the object of serious criticism and re-assessment. What has aroused such skepticism among researchers has been the fact that although first-, second-, and third-generation glutamate receptor antagonists have long yielded promising results in animal models of brain ischemia, they have failed to elicit a neuroprotective action in stroke and traumatic brain injury in humans. Therefore, the theory of excitotoxicity, though a fascinating paradigm, can only

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explain some of the events occurring in the acute phase of anoxic insult but cannot be seen as a major target for developing new therapeutic avenues for brain ischemia. In fact, the energy failure occurring during the course of a stroke episode triggers many events that are strictly dependent on several factors: 1. the type of ischemic event, i.e., focal vs global ischemia 2. the region of the central nervous system (CNS) involved in the mechanism of ischemic damage 3. the distance from the area primarily affected by the ischemic event, i.e., ischemic core vs ischemic penumbra 4. the duration of the ischemic event, and 5. the time elapsing between the onset of the ischemic event and the evaluation of the severity of the insult. Furthermore, the fact that after stroke onset Ca2þ concentrations follow a triphasic response that is not equally sensitive to NMDA receptor antagonists [52, 66] should be taken into account. In fact, during exposure to excitatory neurotransmitters, intracellular calcium increase is followed by a transient return to basal levels. After a free interval of a few hours, a gradually progressing secondary increase occurs. The initial Ca2þ influx can be blocked by NMDA receptor antagonists or by the removal of Ca2þ from the extracellular medium but not by antagonists of non-NMDA receptors [52, 66]. The second delayed Ca2þ increase can be counteracted by extracellular Ca2þ removal but not by the application of an NMDA or a non-NMDA receptor antagonist. Interestingly, this second Ca2þ increase can be accelerated by increasing extracellular Ca2þ or by blocking the plasma membrane of Naþ/Ca2þ exchanger [2, 66]. The changes in [Ca2þ]i probably produce other leak currents that result in irreversible disturbances in ion homeostasis and, eventually, in cell death. Although the dominant mediator of the delayed injury has not yet been established, it is likely that a complex chain of events, including the activation of the Ca2þ-dependent catabolic process, the early damage by Naþ overload that induces oncotic cell swelling and ionic edema, and the dysregulation of the ionic homeostasis of other ions, such as zinc and magnesium, may act to destroy cellular integrity. This indicates that not only glutamate antagonists but also a great variety of other drugs that interfere with the other processes might improve neuronal survival after ischemic damage. In addition, when the features of global and focal ischemia are analyzed in association with glutamate levels, several substantial findings challenge the excitotoxic hypothesis of ischemic injury. First, global ischemia induces a damage that is confined to well-defined vulnerable areas although glutamate is released in the same amount throughout the brain [6, 57]. Second, ischemic damage after global ischemia is delayed for days whereas glutamate levels increase immediately after the onset of the ischemic event. Third, since neuronal death in the core region is mainly due to energy depletion, glutamate toxicity should be considered important only for

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the damage occurring in penumbra, the peripheral region of the ischemic lesion. However, in focal ischemia extracellular glutamate increases up to 100 times above the basal level in the ischemic core, reaches peak values 1–2 hours later, and declines slowly thereafter. By contrast, in the ischemic penumbra, it increases only up to 25 times above the basal level and may return to normal within 30 min even if blood flow does not improve [20]. Fourth, the hypothesis that the possible excitotoxicity is induced by glutamate release is also challenged by the fact that the release of excitatory amino acids can be counteracted by other ischemia-related factors such as acidosis and by the release of hyperpolarizing inhibitory neurotransmitters, which during stroke are released at concentrations higher than those of glutamate [40]. Finally, microdialysis studies of glutamate levels after stroke have clearly demonstrated that although the extracellular glutamate concentration increases sharply and rapidly, reaching concentrations 10–100 times higher than preinsult values [7], this increase lasts only for 10–30 min [7]. Therefore, although glutamate may be involved in the acute neurodestructive phase that occurs immediately after ischemic injury, its normal physiological functions, including the promotion of neuronal survival, are resumed directly after this phase [37]. In addition, delayed treatment with NMDA antagonists suppresses neurogenesis, triggered by focal cerebral ischemia, in the hippocampus [4]. These findings suggest that in addition to damaging neurons immediately after the injury, glutamate may also facilitate repair shortly thereafter. Interestingly, whereas excitotoxic effects of glutamate are short lasting, repair mechanisms appear to be long lasting. The interference with neuronal survival means that NMDA antagonists are unsuitable neuroprotective drugs for stroke therapy. The only way to provide pharmacological neuroprotection with NMDA antagonists would be to administer them before the insult and for a very short period (even minutes) after the injury, circumstances that would be virtually impossible in a clinical emergency setting. Thus, when designing novel therapies, researchers need to consider and respect the physiological role of glutamate in the brain. By focusing on the destructive effects of glutamate after injury and by ignoring its physiological functions, many patients were unnecessarily exposed to glutamate NMDA antagonists. In addition, when we consider that overlooking the potential detrimental role of glutamate has resulted in years of long painstaking but unpromising research, as well as in unprofitable investments from pharmaceutical companies, the need to re-evaluate the physiological role of glutamate and thus to invest human and financial resources in this or other related lines of research becomes apparent. In the last few years, several seminal experimental works have markedly changed the scenario in this field. In fact, it has been shown that some integral plasma membrane proteins, involved in the control of Ca2þ, Naþ, Kþ, and Cl– ion influx or efflux and, therefore, responsible for maintaining the homeostasis of these cations and anions, might function as crucial players in the brain


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ischemic process [1, 43, 50, 64]. Indeed, these proteins may provide the molecular basis underlying glutamate-independent ionic homeostasis dysregulation in neuronal ischemic cell death and, most important, may represent more suitable molecular targets for therapeutic intervention.

3 The Reasons of the Failure of Excitotoxicity Theory In the last decade, several lines of evidence have accumulated against the concept that high levels of extracellular glutamate are associated with neurological disorders and thus may contribute to neuronal death. The reasons for that can be summarized in the following six ‘‘Hossmann postulates’’ [32]: 1. The type of metabolic and biochemical response induced by glutamate exposure is considerably different from that evoked by ischemic injury. In fact, in vitro and in vivo studies clearly demonstrated that a transient suppression of energy metabolism, as well as changes in protein synthesis, occurs after experimental ischemia [19]. In contrast, exposure to a high dose of glutamate changes neither energy state nor protein synthesis rate. Changes in protein synthesis are a consistent and probably fatal event occurring in all kinds of ischemic cell death [31]. Preservation of protein synthesis after glutamate exposure is, consequently, a strong indication that glutamate-induced damage is different from ischemic-induced damage. 2. Microdialysis and other techniques, which allow to measure glutamate levels in the brain after experimentally induced cerebral ischemia, clearly indicate that increases in glutamate after stroke are not necessarily required for induction of the pathological process. In fact, in models mimicking focal cerebral ischemia, threshold determinations of glutamate release clearly show that glutamate rises to high levels in the ischemic core but not in the penumbra [47, 56]. However, the only reason for cell necrosis in the core is energy depletion; therefore, although glutamate can contribute to cell death, it cannot be considered the main player in this mechanism. Furthermore, although the increase in extracellular glutamate is much lower in the penumbra than in the infarct core, a neurotoxic effect cannot be excluded. However, the probability of such an effect is rather small. On the other hand, in global ischemia, the measurement of glutamate in the different brain regions of rats subjected to 4-Vessel Occlusion (4-VO) have revealed that during ischemia the increases in glutamate levels are almost identical to those found in the hippocampus, striatum, cortex, and thalamus [23]. In addition, even within the hippocampus, glutamate release is the same in all affected regions. By contrast, global ischemia induces a region-specific damage and the different hippocampal regions show a selective vulnerability to the global ischemic insult. The difference is even more evident when different durations of ischemia are compared. In fact, despite the varying concentrations of glutamate in the different regions, the pattern of vulnerability does not

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change [24]. This hypothesis is further supported by the fact that ischemia induces glutamate release in regions spared from histopathological damage in the brain [24]. Since neuronal death occurring in the core region is mainly due to energy depletion, glutamate toxicity should be important only for the damage occurring in penumbra. If so, it is possible to hypothesize that glutamate neurotoxicity in vivo is less toxic than in vitro. However, owing to the activation of anaerobic glycolysis, the penumbral region suffers from a substantial degree of tissue acidosis, a condition that has been shown to alleviate glutamate toxicity in vitro [38]. The ischemic penumbra is not affected by its trophic environment because the structural integrity of this region is fully preserved. Therefore, excitotoxicity induced by glutamate is not plausible in this ischemic area. Glutamate toxicity in vitro is a delayed phenomenon that requires 24 hours to for complete evolution [15]. By contrast, the ischemic penumbra survives no longer than 6 hours. At this time point, in fact, the ischemic penumbra becomes the ischemic core [5] Besides glutamate, other ischemia-related factors such as inhibitory neurotransmitters are released and can be expected to reduce excitotoxicity [38, 40, 66]. For instance, GABA is released at the same blood flow level as glutamate and adenosine even at higher flow thresholds. Thus, the relative increase in GABA is more pronounced than that of glutamate [40].

To these six postulates it should be added that, in the whole brain, the level of glutamate can be maintained at a low threshold by all those systems, which being localized on the membrane of neurons and glial cells are able to induce glutamate re-uptake. More important, it must not be forgotten that recent experimental evidence has suggested a protective role for glutamate in the pathophysiology of the ischemic event [37]. In fact, whereas synaptic transmission mediated by NMDA receptors is essential for neuronal survival, blockade of NMDA receptors triggers apoptosis in the developing brain [35, 51]. Environmental enrichment, which stimulates synaptic activity, inhibits spontaneous apoptosis in the hippocampus and is neuroprotective [65]. Accordingly, when NMDA receptor antagonists are administered during slowly progressing neurodegeneration, they markedly exacerbate damage in the adult brain [36]. In addition, NMDA receptor antagonists cause apoptosis in primary hippocampal cultures and can exacerbate apoptosis induced by staurosporine [29]. By contrast, NMDA-receptor-mediated synaptic activation is neuroprotective in vitro and diminishes apoptosis induced by staurosporine [29]. Activation of pro-survival transcription factors, such as cAMP response element-binding protein (CREB), accompanies NMDA-mediated neuroprotection in vitro [29]. In particular, the Ca2þ pool in the immediate vicinity of synaptic NMDA receptors is able to trigger signaling from the synapse to the nucleus via the extracellular signal-regulated kinase (ERK1/2) (Fig. 1) [27]. One important function of this Ca2þ microdomain, which is located near NMDA

Failure of Ionotropic and Metabotropic Glutamate Antagonists Fig. 1 Activation of prosurvival factors by NMDA receptor agonists

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NMDA Channel Activation

Ca2+

CaMK

ERK

P

CBF

P

CREB Nucleus Activation of prosurvival factors

NEUROPROTECTION

receptors, is to prolong CREB phosphorylation induced by synaptic stimulation, thereby enhancing CREB-mediated gene expression. CREB controls transcription of pro-survival genes such as the brain-derived neurotrophic factor (BDNF), the vasoactive intestinal peptide (VIP), bcl-2, and mcl-1 [53, 61]. Thus, the survival-promoting properties of NMDA receptor could derive from the transcription of such pro-survival genes. In addition, NMDA activation induces an increase of cerebral blood flow NO-dependent [22], thus ameliorating brain ischemic conditions. Indeed, neurons in the penumbra that survive ischemic insults are characterized by high concentrations of BDNF, and bcl-2, and activated CREB [60], hence suggesting a sustained induction of pro-survival signals. These findings lead to the logical conclusion that suppression of survival signals promoted by NMDA receptor activation may facilitate cell death. Taken together, there is sufficient evidence to suggest that synaptic activity mediated by NMDA receptors might promote neuronal survival. Blockade of NMDA-mediated synaptic transmission could therefore be detrimental in situations when support by endogenous measures is required, as occurs after stroke or in chronic neurodegenerative disorders. From all the above-mentioned considerations it is possible to state that it is time to conclude that NMDA antagonists

20

G. Di Renzo et al.

have failed and the attention of researchers working in the field has to be redirected to other cation channels, pumps, and ionic transporters.

4 Conclusion: Beyond NMDA Receptors The NMDA receptor has long been viewed as a major player in inducing excitotoxic cell death. For instance, Simon et al. [58] proposed that the inhibition of the receptor would protect neurons from this death. However, further animal trials demonstrated that the neuroprotective effect of NMDA receptor blockers, such as MK-801, was in fact due to a hypothermia induced by the agent and not by a specific action of the drug [9, 10, 11]. From a number of clinical trials, the surfacing of certain adverse effects has deeply discouraged scientists from fulfilling further research goals in the field. Indeed, NMDA antagonists cause a number of physiological perturbations, including an increase in blood pressure, as well as tachycardia [12]. In particular, achieving serum concentrations high enough to equal those neuroprotective ones obtained in rodents has been challenging, for competitive NMDA receptor antagonists cause many toxic side effects when given at the recommended doses [11]. As suggested by Hoyte [34], there is actually ‘‘a loss in translation’’ when moving from animal models to clinical trials. Intriguingly, the latest literature has highlighted that the activation of NMDA receptors can result in either cell survival or cell death depending on whether the receptor is synaptic or extrasynaptic [22, 26, 28, 30]. The activation of synaptic NMDA receptors promotes cell survival by activation of the CaM kinase and Ras–ERK1/2 (extracellular signal-regulated kinase) pathways and subsequent expression of BDNF [28]. In contrast, the activation of extrasynaptic NMDA receptors inactivates the CREB pathway and downregulates BDNF [28]. Beyond the synapse, different glutamate mechanisms might also operate in other parts of the neuron. In particular, in the axon, the release of glutamate and the subsequent activation of AMPA are both initiated by a large Naþ influx and the reverse of Naþ-dependent glutamate transporters [39]. Under some experimental conditions, imbalances in sodium might even be more important than calcium in axonal compartments. In neuronal dendrites, overactivation of NMDA receptors is damaging. However, activation of kainate-type receptors, which are closely related to AMPA receptors, might actually promote growth and remodeling [44]. Ultimately, glutamergic signaling is mediated not just by NMDA and AMPA currents. Many other glutamate receptors and transporters exist in the brain. Careful targeting of other neuronal glutamate receptors and transporters, including the five metabotropic glutamate (mGlu) receptor subtypes and the excitatory amino acid transporter 2 (EAAT2), could also prove fruitful, on the basis of similar variations in their capacity to trigger death or survival [8, 55]. It should be underlined that beyond the glutamateassociated channels, many other ionic channels also carry large ionic currents in


21

damaged neurons [25]. Altogether, it is important to realize that NMDA–AMPA pathways comprise only a subset of the multiple routes of ionic imbalance that are induced in brain injury and neurodegeneration. Another potential limitation of the standard NMDA–AMPA model is the focus on neurons alone. Indeed, some types of glial cells, including astrocytes and oligodendrocytes, play crucial roles in glutamate regulation, and, thus their roles ought also to be considered alongside neurons. More specifically, glutamate uptake by astrocytes via GLAST and GLT-1, the rodent form of the EAAT2, normally keeps extracellular glutamate below toxic levels. If these mechanisms are impaired by ischemia, neuronal excitotoxicity can be amplified [41]. As both astrocytes and oligodendrocytes express NMDA and AMPA/ kainate receptors, they are also vulnerable to high levels of glutamate [18, 41]. But as already seen in neurons, the processes of oligodendrocytes and astrocytes might respond differently from the cell body. It has been proposed that NMDA receptors comprising NR2C and NR3A subunits mediate injury in the glial processes whereas damages to the glial cell bodies are mediated by AMPA/ kainate receptors [63]. Additionally, glia express many different subtypes of mGlu receptors that exhibit a variety of modulatory functions [18]. Because of all these complexities in glial glutamate regulation and signaling, it might perhaps be difficult to design a single NMDA or AMPA antagonist that would protect all neurons and glial cells after stroke. In the final analysis, the standard NMDA–AMPA model of excitotoxicity is perhaps oversimplistic and does not take into account the complex interactions with other parallel routes of ionic entry and imbalance within the injured cell. Accordingly, several other emerging mechanisms of ionic imbalance deserve further attention as we continue searching for neuroprotectants against stroke and brain injury. Some of the potentially promising ones could include sodium/ potassium ATPase, sodium–calcium exchangers (NCXs), sodium–proton exchangers (NHEs), sodium–potassium–chloride (NKCC), Kþ channels, Naþ channels, acid-sensing ion channels (ASICs), transient receptor potential channels (TRPs), and other non-selective cation channels [3]. Finally, we hope that future research projects have to prize glutamate lesson highly in order to be on the straight and narrow path for setting up new effective strategies in stroke therapy.

5 Future Perspectives Two obvious factors emerge from the discussion presented above: (a) Virtually, all preclinical studies have suggested that NMDA antagonists are effectively protective in focal ischemia only if administered immediately after the insult. This property obviously constitutes a clear drawback in using NMDA antagonists in humans, for as highlighted in clinical trials, longer periods of time necessarily elapse between the onset of the insult and the actual administration of the first treatment.

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(b) The possibility exists that NMDA antagonism, though potentially protective in focal ischemia, is also deleterious in that it adversely affects endogenous NMDA-receptor-mediated neuronal-survival mechanisms [33]. However, intriguing futuristic possibilities are receiving attention and new signs of hope appear over the horizon. In fact, although the disappointment regarding the failure of NMDA antagonists is high, the neuroprotective potential of NMDA isoform-specific antagonists acting extrasynaptically remains to be explored. Furthermore, by combining glutamate, non-glutamatergic receptors, pump, and ionic transporter modulators with antioxidants, as well as anti-apoptotic and antiinflammatory agents, it will be possible to cover the time course of stroke development and eventually develop effective treatments for stroke.

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Mitochondrial Channels as Potential Targets for Pharmacological Strategies in Brain Ischemia Rosemary H. Milton and Michael R. Duchen

1 Introduction Ischemic stroke is a major cause of death and disability urgently requiring novel therapeutic approaches. Deprivation of oxygen and respiratory substrate during an ischemic episode has its most immediate impact on mitochondria, the body’s primary oxygen consumers. Mitochondrial function is in turn critically dependent on a range of carriers and channels, most of which remain inadequately understood at the genetic and molecular level. As mitochondrial channels are involved in energy production, calcium handling, generation of reactive oxygen species (ROS) and cell-death related signaling, it is evident that increased understanding of these processes and their regulation will help the search for novel rational approaches to therapy. Most mitochondrial ion channels and transporters have been less extensively characterised than their plasma-membrane-based counterparts, in part because they are more difficult to study directly. Much of the research we discuss here has been undertaken on mitochondria from organs other than the brain; these data must be extrapolated with care. This chapter will focus on the roles of the mitochondrial ATP-sensitive potassium channel (mitoKATP), uncoupling proteins (UCPs) and the permeability transition pore (PTP), although there are also significant but perhaps less intensively studied roles for other mitochondrial channels, transporters, and pumps (see Table 1).

M.R. Duchen (*) Department of Physiology, University College London, London, WC1E 6BT, UK e-mail: [email protected]


27

11q13

6p11.2-q12

Xq24

Uncoupling Protein 2 UCP2

Uncoupling Protein 4 UCP4

Uncoupling Protein 5 UCP5 (or BCMP1)

6 TM helices

6 TM helices

Controversial, reducing ROS production

Controversial, reducing ROS production

Controversial. Regulation of Kþ homeostasis, osmotic pressure and therefore structural integrity Controversial, reducing ROS production

Unknown, maybe plasma membrane KCa channel

Unknown, maybe plasma membrane KCa channel

Mitochondrial Ca sensitive potassium channel mitoKCa

6 TM helices

Controversial. Regulation of Kþ homeostasis, osmotic pressure and therefore structural integrity

Unknown, maybe plasma membrane KATP channel

Unknown, maybe plasma membrane KATP channel

Mitochondrial ATP sensitive potassium channel, mitoKATP

Other UCPs implicated and UCP4 expressed in brain

Other UCPs implicated and UCP4 expressed in brain

Implicated in brain I-R injury

Implicated in heart I-R injury, expressed in brain

Strongly implicated in brain I-R injury, in pre- and postconditioning Opened by cromokalim, pinacidil, diazoxide, BM10195. Blocked by glibenclamide, 5HD Opened by NS1619. Blocked by iberiotoxin ChTx and paxilline. Uncoupling can be simulated with protonophores such as FCCP Uncoupling can be simulated with protonophores such as FCCP Uncoupling can be simulated with protonophores such as FCCP

Table 1 Molecular and functional properties of ionic channels, uniporters, and exchangers in the mitochondria Selected current Channel, pump or Role in mitochondrial Implicated in ischemiapharmacological transporter Gene Structure function reperfusion? tools

[10, 13, 14]

[10–12]

[8–10]

[6;7]

[1–5]

Selected references

28 R.H. Milton and M.R. Duchen

VDAC1 5q31, VDAC2 10q22 VDAC3 8p11.2

SLC25A1 22q11.21, 2 5q31, 3 12q23 4 4q35 22q13.31

Voltage dependent anion channel VDAC

Adenine Nucleotide Translocator ANT

Unknown

Unknown (in mitochondria)

Calcium uniporter

Sodium-calcium exchanger

Translocator Protein TSPO (Peripheral Benzodiazepine Receptor, PBR)

10q21–10q23

Gene

Cyclphilin D CypD

Channel, pump or transporter


Unknown

5 TM helices

Eight b-strands, two a-helices, and one 310 helix Beta-barrel with 13 betastrands and one alpha helix 6 TM helices

Structure

Role in calcium uptake by mitochondria Regulation of ionic concentrations

Putative role in PTP opening

Regulation of metabolism by regulating flux of metabolites, role in opening of PTP Exchanges ADP for ATP, role in opening of PTP

Role in opening of PTP

Role in mitochondrial function

Table 1 (continued)

Implicated in brain I-R injury, implicated at plasma membrane

PTP in general is implicated in brain I-R injury, TSPO has been ascribed a role in PTP opening and specifically in renal ischemia Implicated in heart I-R injury

PTP in general is implicated in brain I-R injury

PTP in general is implicated in brain I-R injury

Strongly implicated in brain I-R injury

Implicated in ischemiareperfusion?

Blocked by ruthenium red and Ru-360 Blocked by CGP37157

PK11195, diazepam, DPA-714

Blocked by bongkrekic acid

Blocked by Cyclosporine A

Selected current pharmacological tools

[25–27]

[23, 24]

[21, 22]

[20]

[19]

[15–18]

Selected references

Mitochondrial Channels in Brain Ischemia 29


Sodium-proton exchanger


Structure Regulation of ionic concentrations

Role in mitochondrial function Implicated in heart I-R injury, implicated at plasma membrane

Implicated in ischemiareperfusion? Blocked by cariporide, amiloride

Selected current pharmacological tools [28]

Selected references

1. Lauritzen, I., De, W., Jr., and Lazdunski, M. (1997) J. Neurochem. 69, 1570–1579 2. Nistico, R., Piccirilli, S., Sebastianelli, L., Nistico, G., Bernardi, G., and Mercuri, N. B. (2007) Int. Rev. Neurobiol. 82, 383–395 3. Bajgar, R., Seetharaman, S., Kowaltowski, A. J., Garlid, K. D., and Paucek, P. (2001) J. Biol. Chem. 276, 33369–33374 4. Gaspar, T., Snipes, J. A., Busija, A. R., Kis, B., Domoki, F., Bari, F., and Busija, D. W. (2008) J. Cereb. Blood Flow Metab. 28, 1090–1103 5. Lee, J. J., Li, L., Jung, H. H., and Zuo, Z. (2008) Anesthesiology 108, 1055–1062 6. Piwonska, M., Wilczek, E., Szewczyk, A., and Wilczynski, G. M. (2008) Neuroscience 153, 446–460 7. Xu, W., Liu, Y., Wang, S., McDonald, T., Van Eyk, J. E., Sidor, A., and O’Rourke, B. (2002) Science 298, 1029–1033 8. Mattiasson, G., Shamloo, M., Gido, G., Mathi, K., Tomasevic, G., Yi, S., Warden, C. H., Castilho, R. F., Melcher, T., Gonzalez-Zulueta, M., Nikolich, K., and Wieloch, T. (2003) Nat. Med. 9, 1062–1068 9. de Bilbao, F., Arsenijevic, D., Vallet, P., Hjelle, O. P., Ottersen, O. P., Bouras, C., Raffin, Y., Abou, K., Langhans, W., Collins, S., Plamondon, J., ves-Guerra, M. C., Haguenauer, A., Garcia, I., Richard, D., Ricquier, D., and Giannakopoulos, P. (2004) J. Neurochem. 89, 1283–1292 10. Pandya, J. D., Pauly, J. R., Nukala, V. N., Sebastian, A. H., Day, K. M., Korde, A. S., Maragos, W. F., Hall, E. D., and Sullivan, P. G. (2007) J. Neurotrauma 24, 798–811 11. Mao, W., Yu, X. X., Zhong, A., Li, W., Brush, J., Sherwood, S. W., Adams, S. H., and Pan, G. (1999) FEBS Lett. 443, 326–330 12. Liu, D., Chan, S. L., de Souza-Pinto, N. C., Slevin, J. R., Wersto, R. P., Zhan, M., Mustafa, K., de Cabo, R., and Mattson, M. P. (2006) Neuromolecular. Med. 8, 389–414 13. Sanchis, D., Fleury, C., Chomiki, N., Goubern, M., Huang, Q., Neverova, M., Gregoire, F., Easlick, J., Raimbault, S., Levi-Meyrueis, C., Miroux, B., Collins, S., Seldin, M., Richard, D., Warden, C., Bouillaud, F., and Ricquier, D. (1998) J. Biol. Chem. 273, 34611–34615 14. Nakase, T., Yoshida, Y., and Nagata, K. (2007) Neuropathology. 27, 442–447 15. Shiga, Y., Onodera, H., Matsuo, Y., and Kogure, K. (1992) Brain Res. 595, 145–148 16. Zhang, W. H., Wang, H., Wang, X., Narayanan, M. V., Stavrovskaya, I. G., Kristal, B. S., and Friedlander, R. M. (2008) Stroke 39, 455–462 17. Korde, A. S., Pettigrew, L. C., Craddock, S. D., Pocernich, C. B., Waldmeier, P. C., and Maragos, W. F. (2007) J. Neurotrauma 24, 895–908 18. Muramatsu, Y., Furuichi, Y., Tojo, N., Moriguchi, A., Maemoto, T., Nakada, H., Hino, M., and Matsuoka, N. (2007) Brain Res. 1149, 181–190 19. Shoshan-Barmatz, V., Israelson, A., Brdiczka, D., and Sheu, S. S. (2006) Curr. Pharm. Des 12, 2249–2270 20. Klingenberg, M. (2008) Biochim. Biophys. Acta

Gene

Channel, pump or transporter

Table 1 (continued)

30 R.H. Milton and M.R. Duchen

Table 1 (continued) 21. Doucet, C., Zhang, K., Desurmont, T., Hebrard, W., Scepi, M., Nadeau, C., Cau, J., Leyre, P., Febrer, G., Carretier, M., Richer, J. P., Papadopoulos, V., Hauet, T., Burucoa, C., and Goujon, J. M. (2007) Nephron Exp. Nephrol. 107, e1–11 22. Papadopoulos, V. (2004) Endocr. Res. 30, 677–684 23. Garcia-Rivas, G. J., Carvajal, K., Correa, F., and Zazueta, C. (2006) Br. J. Pharmacol. 149, 829–837 24. Zhang, S. Z., Gao, Q., Cao, C. M., Bruce, I. C., and Xia, Q. (2006) Life Sci. 78, 738–745 25. Zhang, Y. and Lipton, P. (1999) J. Neurosci. 19, 3307–3315 26. Santo-Domingo, J., Vay, L., Hernandez-Sanmiguel, E., Lobaton, C. D., Moreno, A., Montero, M., and Alvarez, J. (2007) Br. J. Pharmacol. 151, 647–654 27. Motegi, K., Tanonaka, K., Takenaga, Y., Takagi, N., and Takeo, S. (2007) Br. J. Pharmacol. 151, 963–978 28. Toda, T., Kadono, T., Hoshiai, M., Eguchi, Y., Nakazawa, S., Nakazawa, H., Higashijima, N., and Ishida, H. (2007) Am. J. Physiol Heart Circ. Physiol. 293, H3517–H3523

Mitochondrial Channels in Brain Ischemia 31

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R.H. Milton and M.R. Duchen

2 Gene Structure, Structural Features, and Molecular Biology The molecular identity, structural features, and associated molecular biology of most mitochondrial channels remain unknown. All mitochondrial channels and transporters are thought to be encoded by nuclear DNA, however, so their identities should be accessible.

2.1 Mitochondrial ATP-Sensitive Kþ Channel, MitoKATP ATP-regulated Kþ channels, or Kþ flux, have been identified in isolated mitochondrial preparations by several groups. Evidence of a role for mitoKATP in ischemia-reperfusion injury is accumulating (for review see [1]), yet the molecular identity of the channel remains nebulous. While there is some indication that mitoKATP contains the same subunits as the plasma membrane KATP channel, the pharmacology differs (for review see [2]) and the mitochondrial channel has a smaller conductance [3]. The plasma membrane channel is well characterized; it is a hetero-octamer composed of four inwardly rectifying Kþ channels, either Kir6.1 or 6.2, and four sulfonylurea receptor subunits, either SUR1 or SUR2 in various combinations [4]. Kir6.1 and 6.2 and SUR2 have putative mitochondrial targeting sequences within their N-termini, and immunogold electron microscopy and Western blotting have suggested that they can be found in neuronal mitochondria [5]. However, there is doubt about the specificity of commercial antibodies to Kir6.1 [6], and Kir6.2 knockout mice have normal mitoKATP function in cardiac myocytes [7]. It remains possible, therefore, that mitoKATP is composed of entirely different proteins from the surface channel, or the same subunits but with some complex regulation or redundancy.

2.2 Uncoupling Proteins, UCPs In contrast to mitoKATP, the UCPs are a family of proteins with well-established molecular identities. UCPs 2–5 were named in light of their homology with the classical UCP of brown adipose tissue, UCP1, but analogous functions have proved more difficult to demonstrate. Respiring mitochondria export protons across the inner membrane using energy from substrate oxidation, which is ‘‘coupled’’ to the energy required for ATP synthesis, which occurs when these protons re-enter the mitochondria. UCP1 uncouples substrate oxidation from ATP synthesis by providing an alternative route for protons to re-enter the mitochondria (see Fig. 1). UCPs 2 and 3 were identified through their homology with UCP1 in 1997 [8, 9] and UCP4 and UCP5 (BCMP1) were identified in the next 2 years [10, 11]. All UCPs are members of the 35 strong family of mitochondrial anion carriers

Mitochondrial Channels in Brain Ischemia

H+

33

H+ O2* Q

H+ H+

Intermembrane space

C

e-

e-

e-

eMatrix

O2 * + NADH NAD FADH2FAD

O2*

++

HO 4H+ + O2 2 ADP + Pi

I

II

III

H+

IV

H+

ATP

ATP Synthase

H+ H+

Q e-

C e-

e-

e-

+ ++ NADH NAD FADH2FAD

HO 4H+ + O2 2 ADP + Pi

I

II

III

IV

ATP

ATP Synthase Uncoupling

Fig. 1 Mitochondrial respiration. Mitochondrial production of ATP is dependent on the electron transport chain (complexes I-IV, and intermediaries including ubiquinone (Q) and cytochrome c (C)) and the ATP synthase. The electron transport chain transfers redox energy from NADH and FADH2 to oxygen in several stages. As electrons move down the chain, the release of energy is used to transport protons across the inner mitochondrial membrane. The translocation of protons generates an electrochemical gradient expressed primarily as a membrane potential. Protons then move down this electrochemical gradient through the ATP synthase, driving the generation of ATP from ADP and inorganic phosphate. Although electron transfer is generally efficient, electrons may transfer onto molecular oxygen, rather than onto the next stage of the chain, producing superoxide (O2*) which can then contribute to oxidative stress. Uncoupling mitochondria (lower panel) an alternative conductance for protons to re-enter the mitochondria, in competition with the proton path through the ATP synthase. The extrusion of protons by the electron transport chain is therefore uncoupled from ATP synthesis. To maintain the mitochondrial membrane potential and ATP generation, electrons are transferred more rapidly along the electron transport chain, so that the probability of electron leak is reduced. It has been proposed that such a mechanism may reduce oxidative stress.

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and contain three repeats of 100 amino acids, each containing two hydrophobic stretches which correspond to transmembrane helices. UCPs 2, 4 and 5 are expressed in the brain, and may therefore be of relevance to stroke. The ucp2ucp3 cluster in humans is 11q13 between DS11S916 and D11S911 [8]. The fact that the ucp2 gene is only 7 kb downstream from ucp3 possibly reflects a duplication event [12]. The transcription unit of ucp2 is made up of two untranslated exons followed by 6 exons encoding UCP2, which in humans is a 309 amino acid long protein with a mol. wt of 33218. UCP4 was identified through its 34% homology with UCP3, and has been mapped to 6p11.2–q12. Similar to the other UCPs, it has the characteristic 6 transmembrane domains, 3 mitochondrial transporter protein signature motifs, and a putative nucleotide-binding site [10]. UCP5 has 38% homology with UCP2, and is located on the X chromosome in man (Xq25–26) between the genetic markers DXS1206 and DXS1047 [13].

2.3 Mitochondrial Permeability Transition Pore, PTP PTP was first identified by Haworth and Hunter in a series of landmark papers [14–16] as a massive increase in permeability of the mitochondrial membrane following calcium overload and oxidative stress, reflecting the opening of a large conductance channel complex [17]. Until recently, it was held that this channel comprised a multipartite structure with several putative constituent proteins, including the adenine nucleotide translocase (ANT) and the voltagedependent anion channel (VDAC). Recent studies based on inactivation of the genes encoding the candidate proteins [18–20] have cast doubt on the roles of ANT and VDAC, but Cyclophilin D (CypD), the product of the Ppif gene, remains one of the more reliable candidates. The Ppif (peptidylprolyl isomerase) gene consists of 6 protein-encoding exons separated by five introns mapped to 10q21–q23 [21]. Mitochondria from CypD knockout mice do not exhibit typical PTP-like pore opening [18]. While VDAC has long been thought to be a constituent of PTP, mice lacking all 3 VDAC isoforms have now been shown to have intact PTP-like activity [19]. VDAC1 was originally mapped to the X chromosome in the interval Xq13–q21 and VDAC2 to chromosome 21, while VDAC3 maps to chromosome 12 [22]. However, others have mapped VDAC1 to chromosome 5q31 and VDAC2 to chromosome 10q22 [23]. VDAC forms barrels in the outer mitochondrial membrane, consisting of a transmembrane alpha helix and approximately 13 transmembrane beta strands, which enclose aqueous channels. ANT is thought to play a more regulatory role in PTP activity, since mitochondria from mice lacking ANT1 and 2 still exhibit pore opening [20]. The ANT has four isoforms in humans, all consisting of six transmembrane helices, with three repeats of two. Other proteins to have been implicated in PTP function are the peripheral benzodiazepine receptor (PBR; [24, 25]) and the


35

mitochondrial phosphate carrier (PiC; [26]). The complex issue of PTP components and regulators will only be addressed fully when conditional knockout animals or siRNA are used.

3 Tissue and Cellular Distribution MitoKATP is expressed in the inner mitochondrial membrane. In situ hybridization and immunogold electron microscopy [5, 27] have suggested that it is expressed in neuronal mitochondria, but as mentioned, this is not incontrovertible. UCPs 2, 4 and 5 are expressed in mitochondria in the brain, with UCP2, interestingly, being strongly expressed in microglia [28]. The components of the PTP are also present in all cells, although there are variations in susceptibility to opening in different brain areas including throughout the brain [29]. All three VDAC isoforms are present in mitochondria from bovine, rabbit and rat brain [30], while ANT1 is thought to be the most expressed form in the brain [31].

4 Biophysical and Electrophysiological Properties and Regulatory Mechanisms MitoKATP was first described following patch clamping of mitoplasts [3]. The I–V relationship shows that current amplitude increases linearly with hyperpolarization in the negative voltage region and that the linear extrapolation shows a reversal potential at +27 mV, similar to EK which is +29.5 mV. Channel activation is not strongly voltage dependent, however. The conductance described in mitoplasts is consistent with the conductance described in channels reconstituted into artificial membranes [32]. The channels are inhibited by ATP, and activated by GTP and GDP, while modulated by PKC. Most interestingly, mitoKATP is possibly activated by oxidants, as would be generated during ischemia-reperfusion injury [33]. Patch clamping the inner mitochondrial membrane suggests PTP has a conductance of around 1.1 nS. The channel was activated by increased calcium concentrations, but not active in the presence of CsA or at voltages below –40 mV [34]. It has been suggested that the PTP channel has two different conductance states with different selectivities for anions and cations [35, 36] To the best of our knowledge, UCPs have never been subjected to electrophysiological analysis.

5 Physiological Properties MitoKATP is thought to play a role in regulation of mitochondrial Kþ concentration, and therefore mitochondrial volume regulation. Since the enzymes essential for mitochondrial function are present in the mitochondrial

36


membrane, the maintenance of organelle volume and membrane configuration are crucial in respiratory function [37]. ‘Physiological’ functions of UCPs and PTP remain controversial, and both are more frequently spoken of in terms of pathology. UCPs are thought to reduce production of toxic ROS. It has also been proposed that UCPs 2 and 3 are involved in fatty acid export from mitochondria (for review see [38]), in the oxidative metabolism of glutamine [39] and in calcium uptake [40]. PTP is involved in cell death, whereby PTP opening could either dissipate the mitochondrial membrane potential and so impair production of ATP to the extent that the cell undergoes necrosis, or could cause the release of mitochondrial factors such as cytochrome c, which promote signaling pathways causing apoptosis. Less attention has been focused on less devastating roles for PTP; it has been suggested that it releases calcium accumulated by neuronal mitochondria during normal, non-toxic, cellular events [41]. The postulated components of PTP also have important physiological roles; ANT facilitates the exchange of ADP and ATP, while PBR is involved in cholesterol transport [42].

6 Pathophysiological Relevance in Stroke 6.1 MitoKATP Opening mitoKATP channels was initially thought to be protective following association with the phenomenon of ischemic pre-conditioning, wherein a transient and mild ischemia limits the extent of damage incurred following a more severe subsequent ischemia-reperfusion injury. In the heart, the mitoKATP channel openers, cromakalim and pinacidil, affect pre-conditioning and limit ischemiareperfusion injury [43], while physical (ischemic) pre-conditioning is prevented by blockade of mitoKATP channels with glibenclamide or 5-hydroxydecanoate (5HD) [44]. Confirming the pharmacology, it has also been shown that overexpressing Kir6.2 in mitochondria confers protection from hypoxia in HEK and HL-1 cells [45]. It has been argued on the basis of work in the heart that mitoKATP, rather than sarcolemmal KATP channels, is the target of these drugs, but the pharmacological selectivity is not absolute. It is thought, however, that expression of mitochondrial channels is likely to be more relevant in the brain [46]. In the brain, as in the heart, the mitoKATP channel opener cromakalim protects neurons in vitro against both oxygen and glucose deprivation (OGD) and glutamate excitotoxicity [47]. A more mitochondrial-specific opener of KATP channels (BMS 191095) reduced infarct volume in mice subjected to Middle Cerebral Artery Occlusion (MCAO) if given 24 h previously [48]. However, the role of these channels remains unclear as there is some suggestion that blocking, rather than opening, them is protective in hippocampal slices subjected to OGD (using glibenclamide) [49] and in mice subjected to MCAO (using 5HD) [50].


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The prevailing position, however, remains that opening mitoKATP channels prior to an ischemic injury is protective. MitoKATP channel opening could trigger pre-conditioning through the induction of ROS signaling pathways which have downstream effects responsible for protection, but it has also been argued that the channels themselves are the effectors of pre-conditioning and must remain open throughout ischemia (and perhaps reperfusion) to afford protection. It is still not clearly established whether channel openers cause an increase in ROS during pre-conditioning, and if they do, whether these effects are mediated through Kþ or an alternative action of the drug, perhaps directly on the mitochondrial respiratory chain [51–53]. The study most relevant to this chapter, using a mitochondria-specific KATP channel opener on neurons, found that the drug did not increase mitochondrial ROS production while inducing pre-conditioning, but did reduce the levels of ROS production experienced in subsequent glutamate excitotoxicity [54]. Moreover, mitoKATP-opening drugs could exert their effects through uncoupling, reducing ROS production and calcium influx into the mitochondria. Indeed, the mitoKATP channel openers, pinacidil and diazoxide, have mild uncoupling activity [46]. Conversely, it has been argued that the Kþ flux is not large enough to promote uncoupling, and if it were the mitochondria would rupture [55]. Confusingly, it has also been suggested that Kþ ions move in the opposite direction from that required for uncoupling, leaving neuronal mitochondria in the presence of diazoxide [50]. While pre-conditioning has proved a very instructive phenomenon, postconditioning is probably of greater interest to the patient who has just had a stroke. The observation that brief periods of ischemia following an initial and more severe ischemic episode can be protective was initially made in dog hearts [56] and has led to clinical trials in myocardial infarction [57] and replication of the principle in rat brain [58]. From a therapeutic perspective, routine anesthetics seem to offer an opportunity for pharmacological post-conditioning. Administration of isoflurane after ischemia in cortico-striatal slices reduced cell injury in a manner reversible by mitoKATP channel blockers [59]. The positive effects were seen when isoflurane was administered 10 min after OGD; it is obviously important to establish the window within which treatment was effective and examine these principles in vivo. Encouragingly, positive effects of post-conditioning 2 days after injury in rat brain have been reported [60].

6.2 UCPs Just as the endogenous protection afforded by mitoKATP could be exploited therapeutically, so the putative endogenous protective actions of UCPs might also be targeted. There is, however, a conceptual difference, in that the actions of UCPs are more likely to be mimicked than promoted. As mentioned, UCPs are widely believed to reduce ROS production by mitochondria by providing a

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leak pathway to protons, so reducing the mitochondrial membrane potential. Consequently, electrons move along the electron transport chain more rapidly, reducing the probability of electron leak to molecular oxygen to form radicals. However, uncoupling risks compromising ATP synthesis and could only be beneficial to the cell when ‘mild’ [61]. UCP2 is upregulated by pre-conditioning, and infarct volume following MCAO is reduced in mice over-expressing UCP2 [62]. Over-expression of UCP2 was linked to reduced levels of ROS within mitochondria. Interestingly, the converse study, in which UCP2 was knocked out, did not show the converse result. Mice lacking UCP2 are protected from ischemia-reperfusion injury in much the same way that mice over-expressing UCP2 are [28]. The authors attribute this to a compensation effect in these mice, which exhibited increased levels of anti-oxidant expression, again emphasizing the need for conditional knockout mice and siRNA, and conceptually complementary studies. It should also be remembered that other UCPs are expressed, and expressed non-uniformly, throughout the brain. Although there are limited agents available to modulate UCPs, synthetic protonophores exist. Mild uncoupling with the protonophore FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) has been shown to be protective in ischemia-reperfusion injury in the heart and traumatic injury in brain [63, 64], but others have argued that uncoupling is protective neither in cultured neurons [65] nor in synaptosomes [66] since the level of uncoupling required to reduce ROS production causes ATP depletion to an injurious extent. Furthermore, uncoupling is likely to have major systemic side effects, unless it can be targeted to affected tissues, thus it seems that the process and consequences of uncoupling are currently not sufficiently understood to yield major drug targets.

6.3 PTP PTP opening has been most clearly implicated in the heart at reperfusion, which perhaps makes it more amenable to drug therapy than alternative sites, which need to be accessed earlier [67, 68]. The combination of calcium and ROS generation at reperfusion promotes opening of the PTP and cell death through apoptosis (via cytochrome c release) or necrosis. Cytochrome c release via PTP could also be involved in post-conditioning, since it is reduced in post-conditioned brain, relative to ischemic controls [68]. Since PTP activation requires ROS and calcium, it can be modulated through these triggers. Mitochondrially targeted anti-oxidants, such as NAC or SOD mimetics or mito-Q [69] show some promise as neuroprotective agents, but it is not clear if their presence is required before ischemia. One interesting means of reducing ROS involves small Scherzo-Shiller (SS) peptides, which contain ROS-scavenging tyrosine residues. SS-31 is protective against ischemiareperfusion injury in heart and brain [70]. Intriguingly, the authors claim the


39

peptides might exert their effects not only through scavenging ROS, but through acting on PTP directly. Despite the potential problems with peptide treatments, these SS peptides are claimed to be suitable for administration by several routes and cross the blood brain barrier rapidly. PTP opening can also be curbed through limiting calcium overload by blocking the mitochondrial Naþ/Hþ exchanger. One such Naþ/Hþ blocker, cariporide, has been shown to be protective against OGD in neurons [71]. However, clinical trials of cariporide to reduce ischemic events in coronary bypass surgery actually showed these patients were at increased risk of stroke, accentuating the problems of translating principles from isolated preparations to patients, and from heart to brain [72]. The PTP can also be targeted directly. Cyclosporine A (CsA) has long been known to block PTP opening, acting on CypD. CypD is strongly implicated in stroke, since knockout mice have a significantly reduced infarct size following

2 K

1

+

+

H

Outer mitochondrial membrane

H2O MitoKATP

UCP

PTP Inner mitochondrial membrane

CsA

Volume maintained

ROS production decreased

3

Membrane potential maintained, cytochromec release decreased

Fig. 2 Three potential mechanisms of neuroprotection. 1. Opening mitoKATP channels (using cromakalim, pinacidil, BMS191095). The result of this conductance is not clear, but could include maintenance of osmotic pressure, volume and therefore morphology and structural integrity. 2. Increasing proton conductance (mimicking UCPs; using FCCP). Increasing the membrane permeability to protons causes an increase in the speed at which electrons move along the electron transport chain. This reduces the probability of electron transfer to molecular oxygen to produce potentially harmful ROS. 3. Inhibiting PTP opening (using CsA) prevents collapse of the mitochondrial membrane potential and reduces cytochrome c release from mitochondria.

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MCAO [73] and CsA is protective in global cerebral ischemia in rats [74]. CsA is, interestingly, already a widely used immunosuppressant drug, yet it is not simple to apply its usage to stroke patients since it affects all cyclophilins. Nevertheless, it would be interesting to know if transplant patients treated with CsA had reduced risk of ischemia-reperfusion injury. Stavrovskaya and colleagues [75] screened drugs currently approved by the FDA as potential PTP inhibitors and found that promethazine was protective against OGD in neuronal cultures and MCAO in mice. A similar drug, nortriptyline, has now also been shown to have protective effects [76]. Given the roles of several preexisting drugs in blocking PTP, and the development of CsA derivatives such as FR901459 [77] one would hope that the theory could relatively rapidly be transferred to treatments (see Fig. 2).

7 Conclusions Mitochondria contain many proteins and processes which play a central role in defining the progression and outcome of ischemia-reperfusion injury, and so represent valuable potential therapeutic targets. Generally it is difficult to target intracellular channels without affecting processes throughout the cell, and if drugs are required to enter the mitochondria they must contend with the membrane potential [78]. So far, the most productive research on the role of mitochondria in ischemia-reperfusion has been performed in the heart, and heart and brain are not always as closely linked as we would like them to be. These problems, coupled with the general problems of delivering drugs across the blood–brain barrier and treating an acute condition like stroke with enough speed, have led to something of an impasse. However, the central role of mitochondria in ROS signaling, calcium regulation, energy production, and cell death pathways means there is likely to be some aspect of mitochondrial function which can be pharmacologically regulated to offer some hope to stroke patients. A variety of approaches, perhaps reviewing existing drugs with mitochondrial sites of action or perhaps synthesizing new peptides to target mitochondria, coupled with an increased understanding of the identities and functions of the vast range of mitochondrial channels, will hopefully one day lead to increased neuroprotection and preserved neurological function.

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40. Trenker M, Malli R, Fertschai I, Levak-Frank S, and Graier WF. Uncoupling proteins 2 and 3 are fundamental for mitochondrial Ca2+ uniport. Nat Cell Biol 9: 445, 2007. 41. Murchison D and Griffith WH. Mitochondria buffer non-toxic calcium loads and release calcium through the mitochondrial permeability transition pore and sodium/calcium exchanger in rat basal forebrain neurons. Brain Res 854: 139, 2000. 42. Papadopoulos V. In search of the function of the peripheral-type benzodiazepine receptor. Endocr Res 30: 677, 2004. 43. Grover GJ, McCullough JR, Henry DE, Conder ML, and Sleph PG. Anti-ischemic effects of the potassium channel activators pinacidil and cromakalim and the reversal of these effects with the potassium channel blocker glyburide. J Pharmacol Exp Ther 251: 98, 1989. 44. Gross GJ and Auchampach JA. Blockade of ATP-sensitive potassium channels prevents myocardial preconditioning in dogs. Circ Res 70: 223, 1992. 45. Ljubkovic M, Marinovic J, Fuchs A, Bosnjak ZJ, and Bienengraeber M. Targeted expression of Kir6.2 in mitochondria confers protection against hypoxic stress. J Physiol 577: 17, 2006. 46. Bajgar R, Seetharaman S, Kowaltowski AJ, Garlid KD, and Paucek P. Identification and properties of a novel intracellular (mitochondrial) ATP-sensitive potassium channel in brain. J Biol Chem 276: 33369, 2001. 47. Lauritzen I, De Weille JR, and Lazdunski M. The potassium channel opener (–)-cromakalim prevents glutamate-induced cell death in hippocampal neurons. J Neurochem 69: 1570, 1997. 48. Mayanagi K, Gaspar T, Katakam PV, Kis B, and Busija DW. The mitochondrial K(ATP) channel opener BMS-191095 reduces neuronal damage after transient focal cerebral ischemia in rats. J Cereb Blood Flow Metab 27: 348, 2007. 49. Nistico R, Piccirilli S, Sebastianelli L, Nistico G, Bernardi G, and Mercuri NB. The blockade of K(+)-ATP channels has neuroprotective effects in an in vitro model of brain ischemia, Int. Rev Neurobiol 82: 383, 2007. 50. Liu D, Slevin JR, Lu C, Chan SL, Hansson M, Elmer E, and Mattson MP. Involvement of mitochondrial K+ release and cellular efflux in ischemic and apoptotic neuronal death. J Neurochem 86: 966, 2003. 51. Andrukhiv A, Costa AD, West IC, and Garlid KD. Opening mitoKATP increases superoxide generation from complex I of the electron transport chain. Am J Physiol Heart Circ Physiol 291: H2067–H2074, 2006. 52. Facundo HT, Carreira RS, de Paula JG, Santos CC, Ferranti R, Laurindo FR, and Kowaltowski AJ. Ischemic preconditioning requires increases in reactive oxygen release independent of mitochondrial K+ channel activity. Free Radic Biol Med 40: 469, 2006. 53. Minners J, Lacerda L, Yellon DM, Opie LH, McLeod CJ, and Sack MN. Diazoxideinduced respiratory inhibition – a putative mitochondrial K(ATP) channel independent mechanism of pharmacological preconditioning. Mol Cell Biochem 294: 11, 2007. 54. Gaspar T, Snipes JA, Busija AR, Kis B, Domoki F, Bari F, and Busija DW. ROSindependent preconditioning in neurons via activation of mitoK(ATP) channels by BMS-191095. J Cereb Blood Flow Metab 28: 1090, 2008. 55. Garlid KD, Dos SP, Xie ZJ, Costa AD, and Paucek P. Mitochondrial potassium transport: the role of the mitochondrial ATP-sensitive K(+) channel in cardiac function and cardioprotection. Biochim Biophys Acta 1606: 1, 2003. 56. Zhao ZQ, Corvera JS, Halkos ME, Kerendi F, Wang NP, Guyton RA, and VintenJohansen J. Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning. Am J Physiol Heart Circ Physiol 285: H579–H588, 2003. 57. Staat P, Rioufol G, Piot C, Cottin Y, Cung TT, L’Huillier I, Aupetit JF, Bonnefoy E, Finet G, Andre´-Foue¨t X, and Ovize M. Postconditioning the human heart. Circulation 112: 2143, 2005.

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58. Zhao H, Sapolsky RM, and Steinberg GK. Interrupting reperfusion as a stroke therapy: ischemic postconditioning reduces infarct size after focal ischemia in rats. J Cereb Blood Flow Metab 26: 1114, 2006. 59. Lee JJ, Li L, Jung HH, and Zuo Z. Postconditioning with isoflurane reduced ischemiainduced brain injury in rats. Anesthesiology 108: 1055, 2008. 60. Burda J, Danielisova V, Nemethova M, Gottlieb M, Matiasova M, Domorakova I, Mechirova E, Ferikova M, Salinas M, and Burda R. Delayed postconditionig initiates additive mechanism necessary for survival of selectively vulnerable neurons after transient ischemia in rat brain. Cell Mol Neurobiol 26: 1141, 2006. 61. Nicholls DG, Johnson-Cadwell L, Vesce S, Jekabsons M, and Yadava N. Bioenergetics of mitochondria in cultured neurons and their role in glutamate excitotoxicity. J Neurosci Nurs 85: 3206, 2007. 62. Mattiasson G, Shamloo M, Gido G, Mathi K, Tomasevic G, Yi S, Warden CH, Castilho RF, Melcher T, Gonzalez-Zulueta M, Nikolich K, and Wieloch T. Uncoupling protein-2 prevents neuronal death and diminishes brain dysfunction after stroke and brain trauma. Nat Med 9: 1062, 2003. 63. Brennan JP, Berry RG, Baghai M, Duchen MR, and Shattock MJ. FCCP is cardioprotective at concentrations that cause mitochondrial oxidation without detectable depolarisation. Cardiovasc Res 72: 322, 2006. 64. Pandya JD, Pauly JR, Nukala VN, Sebastian AH, Day KM, Korde AS, Maragos WF, Hall ED, and Sullivan PG. Post-Injury Administration of Mitochondrial Uncouplers Increases Tissue Sparing and Improves Behavioral Outcome following Traumatic Brain Injury in Rodents. J Neurotrauma 24: 798, 2007. 65. Johnson-Cadwell LI, Jekabsons MB, Wang A, Polster BM, and Nicholls DG. ‘Mild Uncoupling’ does not decrease mitochondrial superoxide levels in cultured cerebellar granule neurons but decreases spare respiratory capacity and increases toxicity to glutamate and oxidative stress. J Neurochem 101: 1619, 2007. 66. Tretter L and Adam-Vizi V. Uncoupling is without an effect on the production of reactive oxygen species by in situ synaptic mitochondria. J Neurochem 103: 1864, 2007. 67. Gomez L, Thibault H, Gharib A, Dumont JM, Vuagniaux G, Scalfaro P, Derumeaux G, and Ovize M. Inhibition of mitochondrial permeability transition improves functional recovery and reduces mortality following acute myocardial infarction in mice. Am J Physiol Heart Circ Physiol 293: H1654–H1661, 2007. 68. Wang JY, Shen J, Gao Q, Ye ZG, Yang SY, Liang HW, Bruce IC, Luo BY, and Xia Q. Ischemic postconditioning protects against global cerebral ischemia/reperfusion-induced injury in rats. Stroke 39: 983, 2008. 69. Sharma SS and Gupta S. Neuroprotective effect of MnTMPyP, a superoxide dismutase/ catalase mimetic in global cerebral ischemia is mediated through reduction of oxidative stress and DNA fragmentation. Eur J Pharmacol 561: 72, 2007. 70. Szeto HH. Mitochondria-targeted cytoprotective peptides for ischemia-reperfusion injury. Antioxid Redox Signal 10: 601, 2008. 71. Luo J, Chen H, Kintner DB, Shull GE, and Sun D. Inhibition of Na+/H+ exchanger isoform 1 attenuates mitochondrial cytochrome C release in cortical neurons following in vitro ischemia. Acta Neurochir Suppl 96: 244, 2006. 72. Mentzer RM Jr., Bartels C, Bolli R, Boyce S, Buckberg GD, Chaitman B, Haverich A, Knight J, Menasche P, Myers ML, Nicolau J, Simoons M, Thulin L, and Weisel RD. Sodium-hydrogen exchange inhibition by cariporide to reduce the risk of ischemic cardiac events in patients undergoing coronary artery bypass grafting: results of the EXPEDITION study. Ann Thorac Surg 85: 1261, 2008. 73. Schinzel AC, Takeuchi O, Huang Z, Fisher JK, Zhou Z, Rubens J, Hetz C, Danial NN, Moskowitz MA, and Korsmeyer SJ. Cyclophilin D is a component of mitochondrial permeability transition and mediates neuronal cell death after focal cerebral ischemia. Proc Natl Acad Sci USA 102: 12005, 2005.


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74. Akdemir G, Ergungor MF, Sezer M, Albayrak L, Daglioglu E, and Kilinc K. Therapeutic efficacy of intraventricular cyclosporine A and methylprednisolone on a global cerebral ischemia model in rats. Neurol Res 27: 827, 2005. 75. Stavrovskaya IG, Narayanan MV, Zhang W, Krasnikov BF, Heemskerk J, Young SS, Blass JP, Brown AM, Beal MF, Friedlander RM, and Kristal BS. Clinically approved heterocyclics act on a mitochondrial target and reduce stroke-induced pathology. J Exp Med 200: 211, 2004. 76. Zhang WH, Wang H, Wang X, Narayanan MV, Stavrovskaya IG, Kristal BS, and Friedlander RM. Nortriptyline protects mitochondria and reduces cerebral ischemia/ hypoxia injury. Stroke 39: 455, 2008. 77. Muramatsu Y, Furuichi Y, Tojo N, Moriguchi A, Maemoto T, Nakada H, Hino M, and Matsuoka N. Neuroprotective efficacy of FR901459, a novel derivative of cyclosporin A, in in vitro mitochondrial damage and in vivo transient cerebral ischemia models. Brain Res 1149: 181, 2007. 78. Yamada Y, Akita H, Kogure K, Kamiya H, and Harashima H. Mitochondrial drug delivery and mitochondrial disease therapy – an approach to liposome-based delivery targeted to mitochondria. Mitochondrion 7: 63, 2007.

Endoplasmic Reticulum Calcium Homeostasis and Neuronal Pathophysiology of Stroke Alexei Verkhratsky

1 Introduction Endoplasmic reticulum (ER) formed by the endomembrane is arguably the largest intracellular organelle. The ER is present in all types of neural cell, in neurones and in glia [1, 2]. Morphologically, the ER is represented by a system of microtubules and cisternae organised into a complex 3D network. In neurones, the ER extends from the nucleus and the soma to the dendritic arborisation and, through the axon, to presynaptic terminals. Although the lumen of the ER is internally continuous, the spatial segregation of enzymatic systems ascertains remarkable functional heterogeneity of intra-ER compartments. Indeed, the ER is a place where a multitude of critical cellular processes develop. The ER acts both as a cellular synthetic factory, where mRNA is translated into proteins and the latter undergo post-translational folding, and as an intracellular highway where many of these proteins are transported to their final destinations. In addition, the ER is the primary site of formation of phospholipids, glucosylphosphatidylinositols and leukotriens. Besides its synthetic and transporting functions, the ER acts as a complex signalling organelle, able to receive and produce signals, which are of crucial importance for cell activity and survival [3]. In particular, the ER is specifically involved in cellular Ca2+ signalling, being the largest dynamic store of Ca2+ that generates Ca2+ fluxes between the cytosol and the ER lumen in response to extracellular stimulation, thus producing and shaping cytosolic Ca2+ signals [4]. The Ca2+ movements within the ER are also of critical importance [5], as free Ca2+ concentration in the ER lumen ([Ca2+]L) control synthesis and folding of proteins and initiate many of ER output signals. A. Verkhratsky (*) The University of Manchester, Manchester, M13 9PT, UK; Institute of Experimental Medicine, ASCR, 142 20 Prague 4, Czech Republic e-mail: [email protected]


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The ER Ca2+ homeostasis and signalling (Fig. 1) is controlled by several molecular systems, which determine ion fluxes through the endomembrane [1, 4, 6]. These systems are (i) endomembrane Ca2+ pumps of the SERCA (Sarco(Endo)plasmic reticulum calcium ATP-ase) family; (ii) intracellular Ca2+ release channels (which include ryanodine receptors (RyRs), inositol-1,4,5-trisphosphate receptors (InsP3Rs), and may be some other Ca2+ permeable channels such as, e.g. NAADP receptors or TRPV1 channels); (iii) intra-ER Ca2+ buffers and (iv) other ion channels, which maintain the ER membrane potential by counter-balance currents produced by Ca2+ release from the store. The nature of these channels is largely unknown although recently cloned trimeric intracellular cation channels (TRICs) may be responsible for counter-balancing currents carried by monovalent cations [7].

Fig. 1 Molecular physiology of endoplasmic reticulum Ca2+ homeostasis/signalling. Calcium is taken up into the ER by energy-dependent transport accomplished by SERCA, thus building up concentration gradient between the ER lumen and the cytoplasm. Physiological stimulation opens Ca2+ release channels represented by ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (InsP3Rs) localised in the ER membrane. The ER membrane also contains cationic channels, which produce ion fluxes counter-balancing charge redistribution associated with Ca2+ release.

All components of the ER ion moving system are regulated by Ca2+ gradients across the ER membrane in a complex manner [8, 9]. The complement of the Ca2+-regulated channels and Ca2+ pumps converts the ER membrane into the excitable media, which is able to generate propagating waves of Ca2+ channels openings; these openings in turn underlie the well-known phenomena of propagating intra- and intercellular Ca2+ waves. In the following chapters I shall concentrate on the three components of Ca2+ homeostatic/signalling system of the ER, namely on SERCA pumps and Ca2+ release channels of the RyR and InsP3R types.

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2 Gene Structure 2.1 SERCA In vertebrates, the SERCA family is encoded by three distinct genes; each of the three genes has several splice variants. These genes are designated as ATP2A1–3; these produce SERCA1a/b, SERCA2a/b and SERCA3a/b/c/d/e isoforms [10].

2.2 Ryanodine Receptors Ca2+-gated Ca2+ release channels residing in the endomembrane are generally known as ryanodine receptors (RyRs) because plant alkaloid ryanodine was the first specific probe against these proteins. The RyR family is encoded by three major genes, the RyR1, RyR2 and RyR3 [11]; historically these subunits were also known as ‘‘skeletal muscle’’, ‘‘heart’’ and ‘‘brain’’ types, respectively. The RyR genes are very large, and comprise 100 exons. Diversity of RyRs also stems from splice variants of which 13 have been identified [12].

2.3 InsP3 Receptors The InsP3 receptor family comprises three homotetrameric isoforms encoded by distinct genes, InsP3R1, InsP3R2 and InsP3R3 [13]. Further diversity arises from alternative splicing of InsP3R1 (up to six variants) and heteromeric expression [14].

3 Structural Features and Molecular Biology 3.1 SERCA The overall structure of SERCA proteins includes four major domains (see [10] for details). The transmembrane or M-domain contains 10 transmembrane helices, which form Ca2+-binding sites of the pump; other three domains (A, P and N) are cytoplasmic. The A (or actuator) domain and P (phosphorylation) domains are coupled to the M domain whereas the N (or nucleotide-binding domain) is linked to the P domain.

3.2 Ryanodine Receptors Both Ca2+ release channels, the RyRs and the InsP3Rs, are large tetrameric channel proteins, which share a four-clover-leaf-like structure when observed

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by electron microscopy [11]. The RyRs are probably the largest channels discovered so far, the RyR1 monomers comprise 5032–5037 amino acid residues; RyR2 – 4969–4976 residues and RyR3 – 4872 residues, with the mol. wt. of each monomer around 500 kDa [15]. The tetrametic RyR channel complex has two portions: the large (80% of the whole protein) cytosolic (also known as ‘‘head’’) with dimensions of 29 29 12 nm and a central or transmembrane portion (comprised of four transmembrane domains), which extends by 7 nm from the cytosolic domain [16]. This part also forms a central hole of about 2 nm in diameter, where the actual channel pore supposedly resides [17].

3.3 InsP3 Receptors The overall topology of InsP3 receptor is very similar to RyRs; the InsP3Rs are homotetramers, although the monomers are much smaller with mol.wt. around 260 kDa. The InsP3R1 comprises 2749 amino acid residues, the InsP3R2 – 2701 and InsP3R3 – 2671 amino acid residues. The InsP3Rs also have a large cytoplasmic ‘‘head’’-like structure, which contains the InsP3-binding site and a relatively small channel-forming part constructed from six transmembrane domains [13, 18].

4 Tissue and Cellular Distribution 4.1 SERCA The SERCA2b isoform is ubiquitously expressed in all cellular elements of the nervous system, in central and peripheral neurones, in astro- and oligodendrocytes and in microglia [19]. The SERCA 2a and SERCA3 are found almost exclusively in cerebellar Purkinje neurones [1, 19]. The role of SERCA3 isoform remains unresolved; genetic deletion of SERCA3 does not result in any obvious phenotypic change, save minor deficits in relaxation of vascular and tracheal smooth muscles [20]. Within the cells the SERCA pumps are distributed throughout the ER membrane; this distribution can be visualised in living cells by using fluorescent markers such as fluorescent thapsigargin.

4.2 Ryanodine Receptors All three types of RyRs are present in neurones, although expression of the RyR2 subtype dominates. Many neurones co-express either two or three RyR subtypes. The RyR1 is heavily expressed in cerebellar Purkinje neurones whereas RyR3 is found in hippocampal structures, the corpus striatum and the diencephalon [21, 22, 23, 24, 25, 26]. Within the nerve cells the RyRs are


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expressed almost in every part: the RyRs were detected in neuronal somatas, in axons of hippocampal [22], in cerebellar mossy fibres [26] and in presynaptic terminals [27]. RyRs are also heavily expressed in the dendritic tree; their expression is particularly high in dendritic spines of hippocampal CA1 neurones, where they represent the dominant Ca2+ release channel [22]. The expression of RyRs is developmentally regulated. Throughout the embryonic stage, RyR1 mRNA levels are highest in the rostral cortical plate whereas RyR3 mRNA are most prominent in the caudal cortical plate and hippocampus. Relatively low expression of RyR2-specific mRNA was detected in the diencephalon and the brainstem. However, from postnatal day 7 onwards, RyR2 mRNA gradually became the major isoform in many brain regions, while RyR1 mRNA became prominent in the dentate gyrus and in the Purkinje cell layer. Postnatal down-regulation in the caudal cerebral cortex restricted RyR3 mRNA expression to the hippocampus, particularly the CA1 region [28].

4.3 InsP3 Receptors All three subtypes of InsP3Rs were identified throughout the brain, with particularly high expression in Purkinje neurones and in somatas of CA1 cells [18, 22]. The InsP3R1 is the most abundant subtype in the brain whereas InsP3R2 dominates in the spinal cord and is also highly expressed in glial cells. The InsP3R3 are expressed to a much lesser extend; being present in cerebellar granule layer and the medulla [29]. Interestingly, InsP3Rs display cell-specific intracellular distribution. For example, InsP3R1 is found in dendrites, dendritic spines, cell bodies, axons and axonal terminals of cerebellar Purkinje cells, but is mostly confined to somatic regions and proximal dendrites in other neurones [29]. The InsP3R3 is very much concentrated in the neuropil and neuronal terminals [29].

5 Biophysical and Electrophysiological Properties 5.1 Ryanodine Receptors All three RyRs are classical ligand-gated channels, which are activated by Ca2+ from the cytosolic side; the rank order of RyRs sensitivity to [Ca2+]i is RyR1 > RyR2 > RyR3 [30, 31]. The RyRs reconstituted in lipid bilayer membranes and studied under voltage clamp behaved as not perfectly selective Ca2+ channels (PCa2+/PK+ 6–7) with fast activation kinetics (t of activation 0.5–1 ms) and a large conductance (100–500 pS, depending on the nature of ion carrier[16]). Physiological unitary Ca2+ current through RyRs was estimated at 0.5–0.9 pA at 2 mM intraluminal Ca2+ concentration [32].

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5.2 InsP3 Receptors The InsP3 receptors are activated by second messenger InsP3 produced by phospholipase C; this enzyme in turn appears as a part of the phosphoinositide signal transduction cascades, coupled to an extended family of metabotropic receptors abundantly expressed in neural cells. The conductance of InsP3Rs has been estimated at 10–26 pS, being thus considerably smaller when compared with RyRs. The InsP3Rs were also reported to show multiple (up to four) conductance states [33, 34, 35]. The activation of InsP3Rs is synergistically regulated by both InsP3 and cytosolic Ca2+. The most important difference between InsP3R subtypes lies in their different sensitivity to cytosolic Ca2+ concentration: the InsP3R1 has the ‘‘classical’’ bell-shape dependence on [Ca2+]i with a maximal activation at 300–400 nM [33]; the InsP3R2 and InsP3R3 are also stimulated by increase in cytosolic Ca2+, but do not show inhibition at high [Ca2+]i [36].

6 Regulatory Mechanisms 6.1 SERCA Ca2+ ions in the ER lumen act as the main regulator of SERCA pump. Already early experiments on ER vesicles found that an increase in intra-vesicular Ca2+ effectively inhibits Ca2+ uptake [37, 38]. Experiments on cellular preparations when [Ca2+]L was monitored by fluorescent probes clearly demonstrated that a decrease in [Ca2+]L markedly increased the velocity of SERCA-dependent Ca2+ uptake [8, 39, 40]. This peculiar regulation involves interactions between intra-ER proteins calreticulin and Erp57 with SERCA pumps. At high [Ca2+]L the calreticulin–Erp57 complex binds to and inhibits the activity of SERCA whereas lowering of [Ca2+]L results in the protein complex dissociation from the pump, which in turn increased the activity of the latter [41, 42, 43].

6.2 Ryanodine Receptors Cyclic ADP-ribose (cADPR). In nerve cells, cADPR does not open RyRs directly, yet it does potentiate Ca2+-induced Ca2+ release. Intracellular injections of cADPR did not affect resting [Ca2+]i but increase [Ca2+]i transients induced by depolarization [44, 45, 46, 47]. This effect was blocked by high concentrations of ryanodine or by conditioning emptying of the ER stores by incubations with 10–20 mM caffeine, thus suggesting cADPR interaction with RyRs. The cADPR can act as a second messenger, production of which in neural cells could be controlled by muscarinic cholinoreceptors [48], b-adrenoreceprtors [49],


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metabotropic glutamate receptors [50] or NO/cGMP/cGMP-dependent protein kinase cascade [49], although the details of the cADPR-dependent signalling cascades are far from being fully characterized. Nicotinic acid adenine dinucleotide phosphate (NAADP). Although the main action of NAADP is believed to be mediated through specific receptors present in the ER membrane [51], there are indications that NAADP can directly activate RyRs [52,53]. Calexcitin. Calexcitin, a Ca2+ and GTP-binding protein isolated from neurones of conditionally trained Hermissenda [54], is an endogenous modulator of neuronal RyRs and CICR. Calexitin binds to RyRs in a Ca2+-dependent manner and facilitates Ca2+-induced Ca2+ release [55, 56]. Glutamate. Glutamate may influence the RyRs and CICR through direct interactions between metabotropic GluRs and RyRs, which also involved Homer proteins [57]. This mechanism hitherto was experimentally demonstrated in cerebellar granule neurones. In other type of neurones, in cells isolated from the avian nucleus magnocellularis, activation of mGluRs inhibited both RyRs and InsP3Rs by yet unidentified pathway [58]. Neurotrophins. Several neurotrophins were reported to affect Ca2+ release mediated through RyRs in neuronal preparations. For example, NGF was reported to activate RyR-dependent Ca2+ release in cultured cerebellar neurones through activation of p75 receptors [59]. Another neurotrophin, NT-3, potentiated Ca2+ release via RyR/InsP3Rs in neuromuscular junction [60] whereas its cousin NT-4 enhanced CICR in pyramidal neurones from the visual cortex [61]. Finally, neurotrophins may regulate the expression of RyRs; for example NGF markedly up-regulated RyRs in cultured adrenal chromaffin cells [62] whereas BDNF did the same to cerebellar granule neurones [63]. Nitric oxide. Nitric oxide can also act as an endogenous regulator of RyRs [64]. NO activated RyRs in hippocampal CA1 neurones [65]. Activation of RyR-mediated Ca2+ release may also have pathological relevance: for instance NO-mediated Ca2+ release via RyRs could be responsible for Il-1b-induced pyrogenic effects [66]; similarly NO-induced activation of Ca2+ release from a ryanodine-sensitive store can be involved in hypoxic [Ca2+]i rises in brain slices [67].

6.3 InsP3 Receptors Protein kinases and calcineurin. Regulation of InsP3Rs is generally achieved through phosphorylation/dephosphorylation of the receptor molecule. Phosphorylation of the InsP3R, which results in enhanced channel activity, is catalysed by protein kinases A, C and G [68, 69] and by tyrosine kinase [70]. Dephosphorylation of InsP3R reduces the channel activity; this is catalysed by the Ca2+/calmodulin-dependent phosphatase calcineurin [71], which also may regulate the expression of InsP3R at the transcriptional level [72].

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Calmodulin. Calmodulin specifically inhibits InsP3R type 1, which has a respective high-affinity-binding site [73]. Caldendrin. Cytoplasmic Ca2+-binding proteins caldendrins possess dual effect on InsP3Rs. A long-splice variant of caldendrin, when expressed in PC12 or HeLa cells, inhibited IICR triggered by activation of histamine and purinoceptors [74]. Another, shorter splice variant of caldendrin, however, activated the InsP3Rs, was shown to bind specifically to all three InsP3R subtypes. This binding activated InsP3Rs, suggesting the possibility of IICR initiation even in the absence of the natural agonist [75]. Immunophilin FK 506-binding protein 12 (FKBP12). FKBP12 directly associates with InsP3R1; this complex being disrupted upon the addition of the immunodepressants FK506 or rapamycin. Dissociation of InsP3R1–FKBP12 causes an increase of Ca2+ flux through the channel (by increasing channel open time) that can be reversed by adding FKBP12 [76, 77].

7 Physiological Properties 7.1 SERCA The ER plays a dual role in Ca2+ homeostasis and signalling being simultaneously a ‘‘sink and source’’ for Ca2+ ions [78]. This double function is essentially maintained by SERCA pumps, as their activity provide for high intra-ER Ca2+ content, which in turn forms Ca2+ concentration gradient between ER lumen and the cytosol. In turn, the level of [Ca2+]L determines the mode for ER function. Low levels of [Ca2+]L promote Ca2+ buffering whereas the high [Ca2+]L facilitates Ca2+ release [1].

7.2 Ryanodine Receptors The main function of RyRs is to amplify cytosolic Ca2+ signals arising either from plasmalemmal Ca2+ entry or through Ca2+ release via InsP3Rs; this RyR activation is manifested in Ca2+-induced Ca2+ release or CICR [79, 80]. On the molecular level, RyRs, similarly to other ion channels, demonstrate spontaneous openings, which produce elementary Ca2+ release events, generally referred to as ‘‘sparks’’ [81]. These sparks were observed in variety of muscle preparations and were also identified in neuronal cells [27, 82, 83]. Increase in [Ca2+]i or addition of caffeine increases the frequency of sparks until the threshold is reached and regenerative process of CICR ensues. The CICR has been identified and characterised in both peripheral and central neurones (see [1] for review). In various neurones the CICR can be either graded by Ca2+ entry, or can have an ‘‘all or none’’ regenerative behaviour [40, 84]. Importantly, the CICR process, although significantly


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amplifying cytosolic Ca2+ signals produces very minor changes in [Ca2+]L: the free Ca2+ concentration within the ER lumen is rarely reduced by more than 5–10% of the resting level [40]. Regenerative opening of RyRs also underlies propagating ER membrane excitation, which results in formation of intracellular Ca2+ waves [1].

7.3 InsP3 Receptors The InsP3Rs are part of signal transduction cascade, which comprises the plasmalemmal metabotropic receptors (represented by a wide variety of receptors, including mGluRs, P1 and P2Y purinoceptors, muscarinic cholinoreceptors and many more), coupled to phospholipase C (PLC) via G-proteins. Physiological activation of metabotropic receptors by appropriate neurotransmitters results in activation of PLC, that in turn produces second messengers diacylglycerol and InsP3 [85]. In neural cells metabotropic receptors are activated by neurotransmitters and hormones, which results in generation of InsP3induced Ca2+ release or IICR. The IICR in turn can be either spatially localised, which results in generation of microdomains of increased [Ca2+]i, or in certain conditions, trigger global [Ca2+]i response [1]. In neurones local [Ca2+]i microdomains produced by IICR play an important role in synaptic plasticity [86, 87]. In addition to InsP3, the InsP3Rs are regulated by [Ca2+]i and the increase in the latter facilitates the IICR. As a result local increases in [Ca2+]i may trigger Ca2+ release in the presence of constant levels of InsP3 or else assist in recruiting neighbouring InsP3Rs thus creating a propagating Ca2+ waves through the so-called CICR-assisted IICR [1, 88]. Importantly, this property may play a role of coincidence detector, when pairing of metabotropic receptors activation with plasmalemmal Ca2+ entry (resulting e.g. from backpropagating action potentials) synergistically increases the IICR and greatly potentiates Ca2+ signals [88, 89].

8 Pathophysiological Relevance in Stroke Conceptually, ischaemia and stroke trigger dysregulation of Ca2+ homeostasis in neural cells. As a result, cytoplasm became overloaded with Ca2+; an increased [Ca2+]i in turn triggers various death subroutines, which result in neuronal demise via either necrotic or apoptotic way [90]. Part of [Ca2+]i elevation in ischaemic conditions is due to ER Ca2+ release [91, 92]. For example, massive (30 mM) increases in [Ca2+]i following an interruption in blood flow observed in hippocampal neurones were substantially (5 times) reduced by inhibition of the ER with TG [93, 94]. In acute slices prepared from striatum hypoxia induced huge Ca2+ release from the ER [67].

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Similarly, in cultured hippocampal neurones treated with NaCN to mimic anoxic environment, a large [Ca2+]i increase, sensitive to dantrolene and ruthenium red, was observed [95]. Inhibition of RyRs by dantrolene significantly reduced neuronal death in the hippocampus in adult rats, subjected to global cerebral ischaemia [96]. In white matter Ca2+ release from the ER is also involved in producing the ischaemic damage. That is [Ca2+]i increase, which mediates axonal damage, persisted even when Ca2+ ions were removed from the external solutions; at the same time inhibition of RyRs had clear protective effect [97]. Changes in the ER Ca2+ homeostasis and partial depletion of the ER from free Ca2+ may also play an important role in defining the fate of neural cells after stroke and ischaemia. It is well known that decrease in [Ca2+]L can trigger the ER-stress response, which is characterised by an accumulation of unfolded proteins and generation of an array of death signals [3, 98, 99, 100]. The link between ischaemia-induced changes in ER Ca2+ homeostasis and intra-ER chaperone activity in neurones has been identified. It turned out that the oxygen-regulated protein of 150 kDa (orp150) is up-regulated in astrocytes following ischaemic stress, which may explain astroglia resistance to ischaemic conditions. Overexpression of orp150 in cultured rat cortical neurones protected them against ischaemia [101]. Transgenic mice expressing orp150 under the control of a platelet-derived growth factor B-chin promoter displayed high levels of orp150 in the cortex, hippocampus and cerebellum and were much more resistant to global cerebral ischaemia compared to wild-type controls [101].

9 Pharmacological Modulation 9.1 SERCA Pumps The most potent and specific (KD 20 nM), though irreversible SERCA inhibitor is thapsigargin [102]. The cyclopiazonic acid blocks SERCA at concentrations 20–50 mM, and its action is reversible [1].

9.2 Ryanodine Receptors Caffeine. Caffeine is the most popular pharmacological probe for studying RyR-mediated Ca2+ release in cellular preparations. Caffeine rapidly penetrates the plasmalemma, and equilibrates within intracellular compartments. Caffeine stimulates RyRs, at low (1–3 mM) by sensitising the channel to cytosolic Ca2+ whereas at higher (10–20 mM) concentrations caffeine opens the RyR channel and keeps them activated for as long as the drug is present in the system (see [1] for review).


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Ryanodine. Ryanodine action on the RyR is concentration dependent. At low concentrations (1–5 mM), ryanodine promotes RyR channel opening in a sub-conducting (40–60% as compared with a normal) state, which associated with significant (about 20 times) increase in the channel open time. At high concentrations (50 –100 mM) ryanodine completely blocks RyRs [103, 104]. Ruthenium red. Ruthenium red blocks RyRs at concentrations between 1 and 20 mM [105]. At the single channel level, ruthenium red reduces Po by prolonging the closed state of the RyR. It has to be noted that ruthenium red is not very specific as it also inhibits voltage-gated Ca2+ channels, epithelial Ca2+ channels ECaC1, TRPV channels and mitochondrial calcium uptake (see [1] for review). Dantrolene. Dantrolene reduces the open probability of RyRs; the effective drug concentrations are 10–90 mM [106]. General anaesthetics. Halothane and enflurane increase Po of RyR channels, and thus, enhance the Ca2+-induced Ca2+ release [105]. Local anaesthetics. Most of the local anaesthetics, such as procaine, tertracaine, lidocaine, prilocaine, the quaternary amines QX 572 and QX 314 and benzocaine, inhibit RyR-mediated Ca2+ release in millimolar concentrations [105]. Heparin. Heparin stimulates Ca2+ release [107] by increasing channel openings with EC50 0.23 mg/ml in a Ca2+-dependent manner [108, 109].

9.3 InsP3 Receptors Heparin. Heparin blocks the activity of InsP3-gated channels incorporated into lipid bilayers [108] and effectively inhibits IICR in many types of non-excitable and excitable cells, when administered intracellularly. Caffeine. Caffeine at 10–20 mM concentrations is extremely potent inhibitor of InsP3Rs [110]. Direct electrophysiological investigations of purified InsP3Rs demonstrated effective inhibition of unitary InsP3R current. The KD of caffeine action on InsP3R currents was 1.6 and 10 mM completely blocked unitary InsP3R currents [111]. 2-Aminoethoxydiphenyl borate (2-APB). 2-APB inhibits IICR with IC50 40 mM [112, 113]. At the same time, however, 2-APB lacks selectivity as it affects not only IICR but also Ca2+ pumps and store-operated Ca2+ entry [114, 113]. Xestospongines. Xestospongines (Xe’s) belong to a group of macrocyclic bis-1-oxaquinolizidines isolated from the Australian sponge, Xestospongia species [115]. Some of these agents, namely XeA, C and D are membrane-permeable blockers of IICR; of these, XeC was the most potent inhibitor with a KD of 360 nM. Further experiments have demonstrated that XeC blocks [Ca2+]i transients mediated by metabotropic pathways in the PC12 cell line and in primary rat cortical astrocytes. Incubation of cells with 5–20 mM XeC effectively abolished [Ca2+]i responses to bradykinin and carbachol [115]. At the same time XeC was also reported to inhibit SERCA pumps [116].

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Adenophostines. Adenophostines A and B, isolated from metabolites of fungi Penicillium brevicompactum [117], are extremely potent (10–100 times more potent than InsP3) agonists of InsP3Rs [118].

10 Conclusions Endoplasmic reticulum plays a central role in calcium homeostasis and signalling in neural cells. It acts as a dynamic calcium store, able to accumulate, store and release Ca2+ ions in response to physiological stimulation. These main functions of the ER are supported by highly coordinated molecular cascades that produce Ca2+ release (RyRs and InsP3Rs) and Ca2+ uptake (SERCA pumps). Activity of these molecular cascades is regulated by Ca2+ gradients across the ER membrane, which ascertains a great homeostatic potential of the ER-Ca2+-handling system. Intra-ER Ca2+ levels also control the activity of numerous enzymatic processes responsible for synthesis, post-translational modification and trafficking of proteins. Ischaemia and stroke disrupt ER Ca2+ homeostasis, triggering Ca2+-dependent death programmes and initiating ER stress. Although the involvement of the ER in pathophysiology of stroke is generally accepted, the pathological potential of ER Ca2+ homeostatic/signalling cascades remains virtually uncharacterised, while clinical pharmacology of these pathways is nonexistent. Further investigations of pathophysiology of ER Ca2+ homeostasis/signalling systems may open a conceptually new line for developing anti-stroke therapeutic strategies.

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The Naþ/Ca2þ Exchanger: A Target for Therapeutic Intervention in Cerebral Ischemia Lucio Annunziato, Pasquale Molinaro, Agnese Secondo, Anna Pannaccione, Antonella Scorziello, Giuseppe Pignataro, Ornella Cuomo, Rossana Sirabella, Francesca Boscia, Alessandra Spinali, and Gianfranco Di Renzo

1 Introduction The Naþ/Ca2þ exchanger (NCX) belongs to the superfamily of Ca2þ/cation antiporter (CaCA) membrane proteins comprising the following members: (1) the NCX family, which exchanges three Naþ ions for one Ca2þ ion (Reeves and Hale, 1984; Fujioka et al., 2000; Kang and Hilgemann, 2004); (2) the Naþ/Ca2þ exchanger Kþ-dependent family (NCKX), which exchanges four Naþ for one Ca2þ plus one Kþ ion [1, 2]; (3) the bacterial family which probably promotes Ca2þ/Hþ exchange (YRBG) [3]; (4) the nonbacterial Ca2þ/Hþ exchange family (CAX), which is also the Ca2þ exchanger of yeast vacuoles [4]; and (5) the Ca2þ/ cation exchanger family (CCX), which contains the partially characterized molecule previously called Naþ/Ca2þ–Liþ exchanger (NCLX or NCKX6). Among these plasma membrane antiporters, the Kþ-independent NCX is a high-capacity and low-affinity ionic transporter that exchanges three Naþ ions for one Ca2þ ion. When intracellular Ca2þ concentrations ([Ca2þ]i) rise and the cells require the return to resting levels, this exchanger transport mechanism couples the uphill extrusion of Ca2þ ions to the entry of Naþ ions into the cells down their electrochemical gradient. This mode of operation, defined as forward mode or Ca2þ efflux or, more correctly, Ca2þ-exit mechanism, keeps the 104-fold difference in [Ca2þ]i across the cell membrane. In other physiological or pathophysiological conditions when the intracellular Naþ concentrations ([Naþ]i) rise or membrane depolarization occurs, thus reducing the transmembrane Naþ electrochemical gradient, NCX reverses its mode of operation and mediates the extrusion of Naþ and the entry of Ca2þ ions. This mode of operation is defined as reverse mode or Ca2þ-entry mechanism. The mode of operation of the antiporter depends on (1) the Naþ gradient, (2) the Ca2þ gradient, and (3) the membrane potential. Therefore NCX is a major player L. Annunziato (*) Division Pharmacology, Department of Neuroscience, School of Medicine, ‘‘Federico II’’ University of Naples, 80131 Naples, Italy e-mail: [email protected]


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in the regulation of physiological and pathophysiological responses to increases of [Ca2þ]i and [Naþ]i [5, 6, 7].

2 Gene Structure 2.1 NCX Family Three genes coding for the three different NCX1 [8], NCX2 [9], and NCX3 [10] proteins have been identified in mammals. These three genes appear to be dispersed, since NCX1, NCX2, and NCX3 have been mapped in human chromosomes 2, 19, and 14, respectively. The NCX1 gene includes 12 exons spread over a region of about 200 kb; however, the major part of the mature protein is coded by exons 2, 11, and 12. The different brain, kidney, and heart NCX1 promoters are partly located in exon 1, in a region comprised from 80 kb to 20 kb before the coding exon 2. Transcripts deriving from exons 3–8, labeled A through F, are subjected to alternative splicing generating different tissuespecific mature RNAs. In order to maintain an open reading frame, all splice variants must include either transcript of exon 3, named also A, or of exon 4, named also B, which are mutually exclusive in NCX1 mature RNA. At least 15 splice variants have been detected for NCX1, named NCX1.1–NCX1.15. Excitable tissues usually have splicing variants encoded by mRNAs, which include transcript of exon A whereas transcript of exon B predominates in other tissues. The NCX2 gene includes 10 exons spreading on a region of about 150 kb. To date, the promoter sequence and location are unknown and no alternative splicing has been demonstrated for this gene. The NCX3 gene includes nine exons spanning a region of about 150 kb. NCX3 minimal promoter spans for 300 bp, just upstream to exon 1. The exons 2–5 present analogies with exons 2–5 of the NCX1 gene [11]. Transcript regions derived from exons 3, 4, and 5 are subjected to alternative splicing. Thus, NCX3 gene has only three exons, instead of six in NCX1, that participate to alternative splicing of the mature transcript. Exons 6–9 of NCX3 correspond to homologous NCX1 exons 9–12. Six splice variants have been described for NCX3: NCX3.1, NCX3.2, NCX3.3, NCX3.4, NCX3-tN.1, and NCX3-tN.2. The physiological and functional significance of all these alternative splicing forms remains unclear; however, it is possible that tissue-specific pattern of alternative splicing is tailored to meet the specific needs of particular brain regions or neuronal population.

2.2 Gene Transcription Regulation The transcription of NCX1 gene is independent of [Ca2þ]i levels. By contrast, the regulation of NCX2 and NCX3 genes is strictly dependent on this bivalent

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cation, although in opposite directions [12, 13, 14]. Thus, changes in [Ca2þ]i levels in neurons directly regulate the amount of NCX2 and NCX3 produced proteins. The physiological and pathophysiological relevance of this elaborated cellular mechanism to modulate NCX protein diversity is nevertheless not fully understood.

2.3 NCX1 Transcription NCX1 gene has three tissue-specific transcription start sites regulated by heart, kidney, and brain promoters [13, 14]. NCX1 brain promoter is not provided with TATA box sequence, but contains several consensus sequences for specific protein 1 (Sp-1) and three binding sites for activator protein 2 (AP-2). The presence of these three AP-2-binding sites implies that NCX1 brain promoter has a housekeeping role and could also exhibit cell-specific properties similar to those observed for the nerve growth factor gene [15]. In addition, NCX1 brain promoter also contains putative binding sites for ubiquitous transcription factors such as nuclear factor-kB (NFkB) and hypoxia-inducible factor 1 (HIF-1).

2.4 NCX2 Transcription To date, no extensive data are available for NCX2 gene promoter and its transcription regulation. However, it has been demonstrated that NCX2 expression is rapidly downregulated after membrane depolarization in a Ca2þ-dependent phosphatase calcineurin manner [16]. Interestingly, the expression of NCX2 after transient middle cerebral artery occlusion results in a massive downregulation throughout the brain, both at the level of peri-infarct area as well as in non-ischemic surrounding brain regions [17]. These areas are subjected to repetitive spreading depression-like depolarization, originating in the ischemic core and propagating to the surrounding brain regions of the same hemisphere [18]. This depression-like depolarization might be responsible for the observed changes in NCX2 gene expression during ischemia [17].

2.5 NCX3 Transcription The NCX3 minimal promoter contains specific enhancers for both muscle and neuronal expression such as cAMP response element (CRE) sequence [19]. However, the complex constituted by CRE-binding protein (CREB) and CREB-binding protein (CBP), by itself, is not sufficient to initiate the NCX3 transcription activation in vivo, suggesting that other unidentified enhancers might be involved [20]. Interestingly, following permanent occlusion of middle

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cerebral artery in rats, NCX3 transcript increases in the peri-infarct region [17], a territory where pro-survival genes, driven by CRE enhancer, are upregulated [21]. In addition, in primary neuronal cultures, unlike NCX2, depolarization upregulates NCX3 mRNA in a calcineurin-independent manner [16]. NCX3 gene promoter also presents a repressor sequence constituted by a doublet of downstream regulatory element (DRE). This doublet of DRE sequences is bound by a transcriptional factor called Ca2þ-modulated transcriptional repressor downstream regulatory element antagonist modulator (DREAM), whose binding to DNA is inhibited by elevated [Ca2þ]i. When [Ca2þ]i is low, the affinity of DREAM for the DRE sequences increases and consequently NCX3 transcription is inhibited whereas when [Ca2þ]i is high, NCX3 transcription is increased [22]. Thus, the transcriptional regulation of NCX3 is dependent on Ca2þ level. This is a striking example of a feedback regulation of the Ca2þ signal in neurons.

3 Structural Features NCX has nine transmembrane segments [23] that can be divided into an Nterminal hydrophobic domain, composed of the first five TMS (1–5), and into a C-terminal hydrophobic domain, composed of the last four TMS (6–9) (Fig. 1). Amino acid sequence found between TMS2 and TMS3 is called a1 repeat whereas the region between TMS7 and TMS8 is named a2 repeat. Both a1

Fig. 1 Molecular topology of NCX with putative sites were drugs interfere with NCX activity.


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and a2 repeats are located on the extracellular and intracellular space, respectively, and may form a portion of the ion translocation pathway [24, 25]. The N-terminal hydrophobic domain (TMS1–5) is separated from the C-terminal hydrophobic domain (TMS6–9) through a large hydrophilic intracellular loop of 550 amino acids, named the f loop [23]. Although the f loop is not implicated in Naþ and Ca2þ translocation, it is responsible for the regulation of NCX activity elicited by several cytoplasmic messengers and transductional mechanisms, such as Hþ, Ca2þ, and Naþ ions, nitric oxide (NO), phosphatidylinositol 4,5 bisphosphate (PIP2), protein kinase C (PKC), protein kinase A (PKA), phosphoarginine (PA), and ATP. Furthermore, in the f loop, there are two stimulatory Ca2þ-binding domains named CBD1 and CBD2. Each CBD has an immunoglobulin-like fold that binds two Ca2þ ions [23, 26]. CBD1 displays a high affinity for Ca2þ with a Kd of 120–400 nM and is the primary Ca2þ sensor since it is able to detect small increases in cytosolic Ca2þ, thus inducing a large structural changes that activate the exchanger. In contrast, CBD2 displays a low affinity for Ca2þ with a Kd ranging from 820 nM to 8.6 mM and it binds Ca2þ only when [Ca2þ]i is elevated with a consequent modest structural alterations. These two different sensitivity thresholds may enable NCX to function dynamically over a wide range of Ca2þ concentrations and permit high Ca2þ exit or entry in excitable cells. In the C-terminal portion of the f loop, at the level of 562–679 aa, there is a putative sequence responsible for Naþ-dependent inactivation (I1) of NCX. In particular, when [Naþ]i rises above 15 mM this region might bind a 20-amino acid sequence, 219–238, named exchanger inhibitory peptide (XIP), located in the N-terminal portion of the same f loop, causing NCX inhibition [27]. In fact, the mutation of some amino acids of XIP sequence increases or removes NCX Naþ-dependent inactivation [28]. Interestingly, from the pharmacological point of view, an exogenous peptide, having the same amino acid sequence of XIP region in the f loop, exerts a potent inhibitory activity on NCX function [29, 30]. NCX2 and NCX3 proteins consist of 921 and 927 amino acids, respectively, and are characterized by molecular masses of 102 and 105 kDa, respectively. In addition, NCX2 displays a 65% sequence identity with NCX1 whereas NCX3 possesses a 73% sequence identity with NCX1 and 75% sequence identity with NCX2 [24]. All three NCX gene products share the same membrane topology with the amino-terminus located in the extracellular space, and the carboxylterminus is located intracellularly.

4 Molecular Biology NCX1 has an extracellular disulfide bond connecting Cys14 or Cys20, located in the extracellular N-terminal tail with Cys792, present in the loop between TMS6 and TMS7 [31]. Moreover, a1 and a2 repeats are

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speculatively modeled to be a re-entrant membrane loop similar to the poreforming region of ion channels. This arrangement is similar to that of the aquaporin water channels, which have also been found to have intramolecular repeats on opposite membrane surfaces [32]. The center of the hydrophobic segment of a2 repeat has a GIG sequence similar to the GYG motif characteristic of Kþ channel P loops. The expression of a mutated exchanger protein which lacks the C-terminal domain or the N-terminal domain does not show any antiporter activity, suggesting that both domains are required to exert exchanger activity [33].

5 Tissue and Cellular Distribution The NCX1 gene displays an ubiquitous expression and therefore is present in several tissues, including brain, heart, skeletal muscle, smooth muscle, kidney, eye, secretory, and blood cells whereas NCX2 and NCX3 gene products have been found exclusively in neuronal and skeletal muscle tissues [12]. The distribution of the three NCX isoforms in the mammalian brain gave useful insights into the physiological and pathophysiological role played by the different NCX isoforms in the regulation of neuronal function. In fact, NCX1, NCX2, and NCX3 are highly expressed in the mammalian brain, suggesting that NCX proteins may play a role in the regulation of neuronal function. Moreover, NCX1 and NCX3 have several splicing variants that appear to be selectively expressed in different regions and cellular populations of the brain [34, 35]. In cerebral cortex, NCX1 is intensively expressed in the pyramidal neurons of layers III and V within the molecular layer of the cerebral motor cortex. This area, which contains the terminal dendritic field of the pyramidal cells, displays a more intense NCX1 immunoreactivity than that of NCX2. In contrast, the somatosensory cortical area seems to express preferentially NCX2 transcripts. Such anatomical distribution reveals that the upper neurons of the motor system and the terminal neurons of the somatosensory system preferentially express distinct NCX isoforms [36, 37]. Within the hippocampus, the transcripts of the three NCX isoforms display an intense labeling of most neuronal populations. In particular, high levels of the three NCX genes have been detected in the granular cell layers of the dentate gyrus and in the pyramidal cells of CA1, CA2, CA3, and CA4 subfields [37]. The three NCX protein isoforms also display high levels of expression within the hippocampus. Thus, in the orient and radiatum layers of the CA1, NCX3 protein is more intense than NCX1 and NCX2. NCX1 protein expression is particularly intense in the granule cell layer and in the hilum of the dentate gyrus, which constitutes the terminal field of the perforant pathway, the major excitatory input to the hippocampus originating from the enthorinal cortex. In the CA3 area, the NCX1 and NCX3 genes of the mossy fibers projecting from the granule cells located in


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the dentate gyrus are more intense than those of NCX2. This peculiar distribution suggests that distinct NCX isoforms may play a crucial role in controlling the intracellular Naþ and Ca2þ homeostasis of the major afferent, intrinsic, and efferent hippocampal projections. Such circuitries are crucial for the synaptic plasticity phenomena, such as those involved in long-term potentiation (LTP) and long-term depression (Madison et al., 1991). NCX isoforms are also expressed in crucial areas of the extrapyramidal control of motor coordination. In fact, NCX1 mRNA can be detected in the substantia nigra pars compacta, in which dopaminergic cell bodies are localized; the NCX1 protein isoform is present in the striatum, in which the terminal projection fields of dopaminergic nigrostriatal neurons are found. Interestingly, both the transcript and the protein, encoded by the three NCX genes, are abundantly expressed in the nucleus accumbens [36, 37], a brain region involved in the motivational control of motor coordination and damaged following middle cerebral artery occlusion.

6 Biophysical and Electrophysiological Properties Naþ/Ca2þ exchanger has a stoichiometry of the three Naþ for one Ca2þ [38]; recent evidence demonstrated that this ion flux ratio is 3.2 for maximal transport in either direction, and at low rate NCX1 can also transport one Ca2þ with one Naþ ion [39]. The Naþ/Ca2þ exchanger has about a 10-fold lower affinity for Ca2þ than ATP-driven Ca2þ pumps but, when it is fully activated, can have a very high rate of transport with an estimate turnover value comprised between 1000 and 5000 s–1 for the transport of both Naþ and Ca2þ using non-saturating ion concentrations. This value is 10- to 50-fold higher than that showed by ATPdriven Ca2þ pumps [40]. A density of 400 exchanger molecules per square mm of plasma membrane produces a current of 30 pA/F [41]. The direction and the amplitude of the Naþ/ Ca2þ exchanger current depend on (1) cellular membrane potential (VM) and (2) Naþ and Ca2þ gradients. These gradients set the reversal potential of NCX (ENCX) which represents the Vm at which the exchanger reverses its mode of operation. The direction of the NCX current (INCX) depends on the difference between ENCX and the cellular membrane potential. When VM is more negative than ENCX, NCX works in Ca2þ-exit mode, in contrast, when the VM value is more positive than reversal potential ENCX, NCX operates in Ca2þ-entry mode. Since in resting neurons VM is –60 mV and ENCX is –40 mV, NCX operates in Ca2þ-exit mode at a low rate. The amplitude of INCX depends on the difference between VM and ENCX values and on several regulatory factors, such as the presence of inhibitors, phosphorylation, inactivation, and interaction with other proteins.

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7 Regulation of Naþ/Ca2+ Exchanger Activity Several factors are involved in the regulation of Naþ/ Ca2þ exchanger activity: (i) the concentrations of the two transported cations, Naþ and Ca2þ; (ii) the intracellular pH; (iii) the metabolic-related compounds, ATP, PIP2, PKA, and PKC; and (iv) reactive oxygen species (ROS) and reactive nitrogen species (RNS). [Ca2þ]i regulates NCX activity through CBD (see page 69). An increase in [Naþ]i can also regulate the Naþ/Ca2þ exchanger. In particular, when [Naþ]i increases, it binds to the transport site of the exchanger molecule, and after this Naþ influx, an inactivation process of the exchanger occurs. This inactivation process, very similar to the phenomenon occurring in voltage-dependent ionic channels, is named Naþ-dependent inactivation. The intracellular pH can also regulate the exchanger. [Hþ] strongly inhibits NCX activity under steady-state conditions, in fact, reductions in pHi values, as little as 0.4, can induce a 90% inhibition of NCX activity. Such inhibitory action depends on the presence of intracellular Naþ ions; hence, the action exerted by Hþ ions is pathophysiologically relevant with regards to brain and heart ischemia. ATP, acting as a phosphoryl donor molecule, may increase the activity of the exchanger in a number of ways. First, by activating G-protein-coupled receptors, via endogenous and exogenous ligands. Second, ATP can stimulate the activity of the Naþ/Ca2þ exchanger through the pathway involving PKC or PKA activation, and each of the NCX isoforms has distinctive putative phosphorylation sites. Finally, the other mechanism by which ATP can activate NCX occurs through the production of the lipid PIP2. In fact, this lipid binding the XIP region of the f loop eliminates NCX inactivation and stimulates NCX function. Interestingly, ATP cellular depletion interferes differently on the three isoforms, as it inhibits NCX1 and NCX2 but does not affect NCX3 activity [42, 43]. Among the regulatory factors, Naþ/Ca2þ exchanger is sensitive to reactive oxygen species, since modifications of the cellular redox state can cause an increase of NCX activity [5].

8 Physiological Properties The Naþ/ Ca2þ exchanger protein is present in both neurons and glia, and may play a relevant function in different neurophysiological conditions. In neurons, the level of expression of NCX is particularly high in those sites where a large movement of Ca2þ ions occurs across the plasma membrane, as it happens at the level of synapses [36, 44]. Specifically, during an action potential or after glutamate-activated channel activity, Ca2þ massively enters the plasma membrane. Such phenomenon triggers the fusion of synaptic vesicles with the


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plasma membrane and promotes neurotransmitter exocytosis. After this event, outward Kþ currents repolarize the plasma membrane, thus leading to VGCC closure. According to the diffusion principle, Ca2þ ions are distributed in the cytosolic compartment, reversibly interacting with Ca2þ-binding proteins. Residual Ca2þ is then rapidly extruded by the plasma membrane Ca2þ ATPase and by NCX. The Naþ/Ca2þ exchanger becomes the dominant Ca2þ extrusion mechanism when [Ca2þ]i is higher than 500 nM, as it happens when a train of action potentials reaches the nerve terminals. In glial cells the Naþ/Ca2þ exchanger is also significantly expressed [45, 46, 47, 48], but the precise role of the exchanger has not yet been determined. To characterize the precise role of the different NCX genes, knockout mice have been generated lacking NCX1 or NCX2 or NCX3. Targeted deletion of NCX1 results in NCX1-null embryos that die before birth [49]. Mice lacking NCX2 gene, the major isoform expressed in the brain, exhibit an enhanced performance in several hippocampus-dependent learning and memory tasks, together with a significantly delayed clearance of elevated Ca2þ following depolarization [50]. In addition, the frequency threshold for LTP and longterm depression in the hippocampal CA regions was shifted to a lowered frequency favoring LTP in these knockout mice [50]. Interestingly, mice lacking ncx3 gene exhibit reduced motor activity, weakness of forelimb muscles, and fatigability in comparison with ncx3+/+ mice [51]. However, since NCX3 is expressed in the CNS and in peripheral nervous system, it cannot be established whether these symptoms can be attributed to CNS defects or to alterations at the neuronal muscular junctions and skeletal fibers levels.

9 Pharmacological Modulation More than 40 years ago, amiloride and amiloride analogs were described by Cragoe [52], by a biological screen process, as potent inhibitors of sodium channels in urinary epithelium thus acting as a potassium-sparing diuretics. Amiloride and its analogs were subsequently shown to be inhibitors of other membrane transporters. In the same years of amiloride synthesis, Baker and Blaustein [53] functionally discovered the existence of the ubiquitous plasma membrane NCX. Amiloride, at very high concentrations, was found to be an effective inhibitor of NCX. Since then, amiloride has been used by numerous investigators, as a probe to block NCX function [54]. Unfortunately, two major drawbacks have limited its use. First, millimolar concentrations were required for its NCX inhibitory activity; second, it lacked of specificity, for it can also inhibit both the epithelial Naþ channels (ENaC) at micromolar concentrations [55] and the Naþ/Hþ exchanger (NHE) in the millimolar range. In the effort to develop amiloride derivatives provided with greater selectivity, Cragoe synthesized compounds more specific for NCX and NHE, respectively. In particular, two classes of amiloride analogs have been developed. The amiloride analogs of

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the first class, such as 5-[N-methyl-N-(guanidinocarbonylmethyl)] amiloride, bear substituents on the 5-amino nitrogen atom of the pyrazine ring [56]. They lack inhibitory properties on ENaC and NCX, even though they display greater effectiveness in inhibiting NHE in the 1–10 mM range [57, 58, 59]. The compounds of the second class, having no inhibitory effect on the Naþ/Hþ exchanger, bear substituents on the terminal guanidino nitrogen atom and behave as specific inhibitors (Ki 1–10 mM) of ENaC and NCX. Among these compounds, CB–DMB appears to be the most specific inhibitor of NCX activity, for it has no inhibitory properties against NHE and the ENaC in excitable cells [54], such as neurons, in which the kidney ENaC are not expressed [56, 57] (Table 1). Table 1 Inhibitors and activators of NCX activity Pharmacological class Compond NCX Inhibitors 1. Amiloride Derivatives 2. Substituted Pyrrolidines 3. Isothiourea Derivatives 4. Ethoxyanilines 5. Benzofuran Derivatives 6. Quinazolinone Derivatives 7. Thiazolidine Derivatives 8. Phenoxypyridine Derivatives 9. Nicotinamide Derivatives 10. Piperidine Derivatives 11. Ylacetamide Derivatives 12. Benzyloxyphenyl Derivatives 13. Peptides 14. Small Interference RNA 15. Antisense Oligodeoxynucleotides 16. Inorganic Cations NCX Activators 1. Redox Agents 2. Organic Compounds 3. Inorganic Cations 4. Agonists of GPCRs 5. Peptides

CB-DMB, DCB, DMB Bepridil KB-R7943 SEA0400 Amiodarone SM-15811 SN-6 JP11092454 6-[4-[(3-fluorobenzyl)-oxy]phenoxy]nicotinamide YM-252077 YM-270951 YM-244769 XIP, Glu-XIP, FMRFa, FRCRCF siRNA-NCX1, siRNA-NCX1, siRNA-NCX3 AS-NCX1, AS-NCX2, AS-NCX3 Ni2þ, La2þ, Gd2þ DTT, GSH, Fe2þ, O2 and of Fe3þ, H2O2 Diethylpyrocarbonate (DEPC) Liþ a, b Agonists, Histamine, 5HT2c Agonists, Endothelin-1, Angiotensin-II Insulin, Concanavalin A

More recently, Shigekawa’s group screening a compound library for the inhibitions of Naþ-dependent Ca2þ uptake identified an isothiourea derivative named KB-R7943 which gained the reputation as a specific compound for inhibiting the antiporter and therefore as a probe to test NCX functional role [60]. KB-R7943 in the low micromolar range, is able to block the reverse mode


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operation of the antiporter whereas concentrations 30 times higher are needed to inhibit the forward mode [61, 62]. However, recent reports have obscured its reputation as a selective probe for NCX inhibition. Indeed, several studies showed that KB-R7943 also exerts an inhibitory effect on several other pharmacological targets such as NHE, dihydropyridine (DHP)-sensitive Ca2þ uptake, passive Naþ uptake, Ca2þ-ATPases and Naþ, Kþ-ATPase [59], and receptoroperated NMDA channels [62]. In addition, it has been reported that the NCX inhibitor KB-R7943 activates large-conductance Ca2þ-activated Kþ channels in endothelial and vascular smooth muscle cells [63]. On the other hand, in the last decade several studies reported CB–DMB as an effective probe to study the role of NCX in some pathophysiological mechanisms including platelet hyperactivity in diabetes [64], collagen-induced platelet activation [64], and in vitro and in vivo ischemic cerebral death mechanisms [30, 65, 66, 67, 68]. In 2001, a new compound belonging to the ethoxyanilines family, SEA0400, was reported as being the most potent NCX inhibitor available at the time, IC50 = 5–92 nM, with a predominant activity on NCX1, a lower affinity for NCX2 and no effect on NCX3. However, the specificity of SEA0400 on NCX activity has recently been questioned, since it can also interfere with Ca2þ movement across the cell membrane. In 2002, by screening benzyloxyphenyl derivatives, Iwamoto’s group discovered the new compound SN-6, which differs from KB-R7943 only in the substituent of phenyl moiety. The presence of this phenyl group confers to this antiporter inhibitor a more selective action on NCX1 rather than NCX3. Using chimeric analysis and subsequent site-directed mutagenesis some critical amino acid residues responsible of SN-6 inhibition in the XIP region of the antiporter have been identified. Interestingly, SN-6 preferentially acts on the exchanger under ATP-depleted conditions. Recently, the further screening of new benzyloxyphenyl derivatives revealed a highly potent NCX inhibitor, named YM-244769. This orally bioavailable compound is more potent in inhibiting NCX3 than NCX1 and NCX2 in the reverse mode, but it is not active on the forward mode of operation of the three antiporter isoforms [69]. Recently, two more selective strategies able to inhibit each of the three NCX gene products, has been introduced as pharmacological tool. These strategies consist in the antisense oligodeoxynucleotide technique and small interference RNA directed against mRNA sequence of NCX1, NCX2, and NCX3 [70, 71]. With this potentially therapeutic approach it has been possible to discriminate the role played by each of the three NCX gene products in preclinical models of pathological states in which this antiporter is involved. The availability of pharmacological agents capable of stimulating the activity of NCX, either in the reverse or in the forward mode of operation, may represent a useful strategy to adopt in some pathophysiological conditions, such as brain ischemia. The pharmacological stimulation of the antiporter could, in fact, contribute to the re-establishment of intracellular Naþ and Ca2þ ion homeostasis. Among NCX activators there are monovalent cations such as Litium that stimulates the Naþi-dependent Ca2þ uptake of all three NCX gene products with low affinity. Reducing agents such as GSH, DDT,

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Fe2þ, and O2– superoxide in presence of oxidating agents such as Fe3þ, H2O2, GSSG, and O2 are able to stimulate NCX activity [72]. Organic compounds, such as agonists of G-protein-coupled receptors, diethylpyrocarbonate, concavallin A, NGF, and insulin, are capable to enhance NCX activity at protein level by biochemical modifications or at transcriptional level by regulating the gene expression of NCX1, NCX2, and NCX3 (Fig. 2).

Fig. 2 PI3-K/Akt1 transductional pathway activated by NGF acting on ncx1 gene expression and on NCX3 post-transductional regulation.

10 Pathophysiological Relevance in Stroke 10.1 NCX Gene and Protein Expression During Ischemia During focal ischemia NCX gene products are differently regulated in the ischemic core, in the peri-infarct area, as well as in intact brain regions. After permanent middle cerebral artery occlusion (pMCAO) all three transcripts NCX1, NCX2, and NCX3 are downregulated by 90% in the ischemic core, including prefrontal cortex (PFC) and part of the striatum, although NCX2 reduction occurs earlier [17]. In the other brain regions, belonging to the peri-infarct zone and including the infralimbic and prelimbic cortices and in the tenia tecta, NCX1 and NCX3 mRNAs display an upregulation. By contrast, in the same subregions, pMCAO elicited a mitigated decrease in NCX2 mRNA expression. In conclusion,


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pMCAO seems to affect NCX gene expression in a differential manner, depending on the exchanger isoform and brain regions involved in the insult. Interestingly, the pattern of expression of these three NCX transcripts mirrors the decline of the respective proteins in the core region [71]. The upregulation of NCX3 that occurs in the peri-infarct region, as opposed to NCX2 downregulation, has been interpreted as a compensatory mechanism induced by NCX3 isoform to counterbalance the reduced activity of the NCX2 protein thus counteracting the dysregulation of [Naþ]i and [Ca2þ]i homeostasis. Evidence for NCX3 neuroprotective role relies in the remarkable broadening of the infarct volume occurring when NCX3 protein is knocked down with a selective antisense oligonucleotide, thereby worsening the neurologic deficits [71]. Accordingly, it has been recently showed in ischemic NCX3–/– mice that NCX3 exerts a neuroprotective effect [73]. The discrepancy of NCX2 and NCX3 expression in response to stroke suggests, once again, that when NCX2 expression decreases in response to hypoxic conditions, NCX3 may assume a replacing function. Consistently, biochemical studies have clearly demonstrated that whereas NCX2 activity is strictly dependent on ATP levels, which are lowered during the development of brain ischemia, NCX3 is the only NCX gene product that is ATP independent [42, 43].

10.2 NCX Activity During In Vitro Anoxia Recent studies performed with the help of cloned cells stably expressing each isoform of the Naþ/Ca2þ exchanger, demonstrate that BHK cells transfected with the brain-specific NCX3 gene (BHK–NCX3), unlike NCX1(BHK–NCX1) and NCX2- (BHK–NCX2) transfected cells, are able to maintain [Ca2þ]i homeostasis during hypoxia plus re-oxygenation-induced Ca2þ overload, although ATP concentrations are reduced. In fact, hypoxia plus reoxygenation induces a lower increase of [Ca2þ]i in BHK–NCX3 than in BHK–NCX1 and BHK–NCX2. In addition, BHK–NCX3 are more resistant to chemical hypoxia plus re-oxygenation than BHK–NCX1 and BHK–NCX2. Interestingly, such augmented resistance can be counteracted by the selective silencing of the NCX3 isoform and by amiloride-derivative inhibitor of NCX, CB–DMB. The peculiar capability of NCX3 isoform to maintain [Ca2þ]i in the physiological range during hypoxic conditions might be correlated with its ability to work in the forward mode of operation in the presence of reduced ATP levels. In this regard, it has been reported that the NCX isoforms display a different sensitivity to ATP levels in the reverse mode of operation [43, 74]. Moreover, chemical hypoxia plus re-oxygenation produces a loss of mitochondrial membrane potential (Dc), a parameter related to the mitochondrial calcium homeostasis, in BHK–NCX1 and BHK–NCX2, but not in BHK–NCX3.

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Altogether, these results indicate that the NCX3 isoform, which is selectively expressed in the brain, more significantly contributes to the maintenance of [Ca2þ]i homeostasis during experimental conditions mimicking ischemia, thereby preventing mitochondrial Dc collapses and cell death. On the other hand, if anoxia is produced directly in primary cortical neurons, by means of oxygen glucose deprivation (OGD), the expression and the electrophysiological activity of the ubiquitous isoform NCX1 increases via nuclear factor kappa B (NF-kB). This increased activity seems to exert a neuroprotective role since NCX1, working in reverse mode of operation, promotes Ca2þ refilling into ER, thus preventing store depletion and consequent ER stress, which occurs after OGD [75].

10.3 Effect of Pharmacological NCX Modulation on Brain Ischemia In in vivo models, reproducing human cerebral ischemia through the occlusion of the middle cerebral artery, the inhibition of NCX, induced by Bepridil and by the selective amiloride derivative inhibitors CB–DMB [30], aggravates brain infarct whereas the activation of the antiporter with redox agents reduces the cerebral infarctual area [71]. At variance with these data, the inhibition of NCX with the isothiurea derivative KB-R7943 and SEA0400 reduces brain injury in the model of transient middle cerebral artery occlusion [76]. However, KB-R7943, besides blocking the antiporter, also produces a remarkable and prolonged hypothermic effect [71] that exerts, by itself, a relevant neuroprotective action in cerebral ischemia. On the other hand, by inhibiting other cellular ionic transport mechanisms and receptors, such as NMDA receptors and L-type Ca2þ channels [76], the same drug may yield a neuroprotective effect (Table 2). An explanation of the worsening effect exerted by NCX inhibitors relies on the different mode of action of this antiporter in the ischemic core and in the penumbra regions (Fig. 3). In particular, it is conceivable that, since in the penumbral region pump ATPase activity is still preserved because ATP levels are partially affected, NCX may likely operate in the forward mode. As a result, by extruding Ca2þ ions, the exchanger favors the entry of Naþ ions. Therefore, the pharmacological inhibition of NCX in this area reduces the extrusion of Ca2þ ions, thus enhancing Ca2þ-mediated cell injury. In contrast, in the ischemic core region, in which ATP levels are remarkably low and Naþ/Kþ ATPase activity is completely compromised, intracellular Naþ ions massively accumulate because of Naþ/Kþ ATPase failure. Hence, the intracellular Naþ loading promotes NCX to operate in the reverse mode as a Naþ efflux–Ca2þ influx pathway. This modality of operation may help brain cells to survive to the excessive [Naþ]i and water overload [71]. In conclusion, the NCX pharmacological inhibition in this core region further worsens the necrotic lesion of the

8.1

7.3

1–2.5 >30 0.05–0.09

0.3–30

0.1–1

Bepridil

CB-DMB

KB-R7943

SN-6

Glu-XIP

SEA0400

IC50 (mM)

Drug

Forward > Reverse mode

Forward > Reverse mode Forward > Reverse mode Forward > Reverse mode Forward > Reverse mode Reverse mode

Mode selectivity

Ala809, Val820, Gln826, Gly833, Asn839 Phe213, Gly833, Tyr224, Tyr226, Tyr228, Tyr231 Phe213, Gly833 and Asn839, Val227 and Val238 445–455

Amino acid regions

TMS5, a-2 repeat, XIP region, a-2 repeat f loop

a-2 repeat

Naþ-binding site

f loop

Site of action

Table 2 Molecular pharmacology of NCX inhibitors

NCX1, NCX2, NCX3

NCX1 > NCX3 > NCX2

NCX1 > NCX2

NCX1 = NCX3 > NCX2 NCX3 > NCX2 > NCX1

Isoform specificity

The Naþ/Ca2þ Exchanger 79

80


Fig. 3 Hypothetical mode of operation of NCX in the penumbra and core regions of ischemic brain.

surviving glial and neuronal cells whereas the activation of NCX contributes to ameliorate ischemic damage.

10.4 Effect of NCX Knocking-Down and Knocking-Out on Brain Ischemia In rats bearing pMCAO, oligodeoxynucleotides blocking NCX1, NCX2, and NCX3, can induce different effects on the development of brain ischemia [71]. In fact, after pMCAO, the neuroprotective effect exerted by NCX is prevalently due to NCX1 and NCX3 products [71]. To determine the precise role of the different NCX genes in brain ischemia, genetically modified mice, lacking NCX1 or NCX2 or NCX3 have been generated. Unfortunately, targeted deletion of NCX1 results in NCX1-null embryos that do not have a spontaneous beating heart and die in utero. Studies conducted in NCX1 heterozygous mice that survive until adultness, showed that these animals did not exhibit increased vulnerability to ischemic damage, despite a reduction of NCX1 protein by almost 50% and of NCX activity by 60% [77]. On the other hand, mice deficient for NCX2 in the brain exhibit a worsening in the ischemic brain lesion induced through transient occlusion of MCA [78]. In in vitro ischemia, a significantly slower recovery in population spike amplitudes, a sustained elevation of [Ca2þ]i and an increased membrane


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depolarization were developed in the NCX2-deficient hippocampus [78]. The third isoform of the antiporter, NCX3, which is expressed selectively in the brain and in the muscle and is able to work also in absence of ATP, plays a relevant role in the ischemic process. In fact, it has been recently shown that ncx3 gene ablation leads to a worsening of brain damage after focal ischemia [73]. In addition, cortical neurons and hippocampal organotypic cultures from ncx3–/– mice display a massive neuronal death when exposed to OGD plus re-oxygenation [73].

11 Therapeutic Perspectives Nevertheless, the great effort to find new and more effective therapeutic interventions in ischemic stroke, the only available pharmacological treatment remains the administration of rTPA that must be administered within the therapeutical window of 3 h from stroke onset. The rationale of targeting NCX activity and/or expression lies on the possibility to pharmacologically modulate this crucial regulator of intracellular Ca2þ and Naþ homeostasis, in order to help neurons and glial cells to survive in the critical penumbra area and to save the major number of cells in the ischemic core. Indeed, most of the preclinical studies specifically targeting NCX isoforms and performed with cellular and animal models reproducing human ischemia suggest that an enhancement of the expression and the activity of CNS NCX isoforms may contribute to reduce the ischemic damage. In fact, their inhibition increases brain vulnerability to the insult. In the last 40 years, most of the medicinal chemistry studies have been devoted to the development of compounds inhibiting global NCX activity in the hope of obtaining compounds active in heart ischemia. However, the new data obtained in cerebral ischemia suggest that pharmacological activators rather than NCX inhibitors may help neurons and glial cells to overcome the stroke-mediated ionic dysregulation. This strategy may be pursued acting at the protein, transductional or transcriptional levels (Fig. 4). First, at protein level, it would be conceivable to identify a functional moiety of drugs working as selective activator of those cerebral NCX isoforms involved in the process of brain damage. This could be achieved by means of quantitative structure relationship activity approach starting from the molecular structure of already available NCX antagonists. Another approach could be pursued by developing cargo compounds consisting of a carrier able to cross the blood–brain barrier and of a molecule provided with neurotrophic factor properties, such as NGF. This new chemical entity could activate NGF/TrkA/Shc/PI3-K/Akt transductional pathway in the brain by stimulating tyrosine kinase receptors, thus inducing an increased expression and activity of NCX1 and NCX3 isoforms [67].

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Fig. 4 Potential steps in the pharmacological regulation of NCX isoform expression and activity.

12 Conclusions An increasing amount of evidence seems to suggest that NCX plays a pivotal role in maintaining intracellular Naþ and Ca2þ homeostasis during pathophysiological conditions in the brain. Indeed, it still remains to be fully clarified whether it is the suppression or the activation of the exchanger that yields potentially beneficial effects on a number of neurodegenerative diseases, such as ischemia, Alzheimer’s disease, aging, and white matter trauma. A number of conflicting results on the modulating effects of the exchanger have highlighted such differences. For instance, the severity and extension of brain injury may vary depending on whether the exchanger is activated or inhibited. Specifically, in animal models of cerebral ischemia, consequent to permanent vascular occlusion, the pharmacological activation of NCX reduces brain damage whereas drugs provided with inhibitory properties aggravate the infarct lesion as well as transgenic mice lacking ncx2 or ncx3 gene. Data derived from drug strategy and knockout mice seem to indicate that the possible development of agents able to selectively activate each of the three NCX gene products might achieve a more promising therapeutic goal. Unfortunately, although efforts to synthesize organic compounds that target the different NCX gene products have been made, no highly specific agents have yet been available. A further obstacle underlying the pharmacological modulation of brain NCX activity is represented by the difficulty of compounds to cross the blood–brain barrier. In this regard, promising strategies entail the conjugation of organic and


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peptidergic drugs with carrier molecules that are substrates for transporters present on the cell membranes of blood–brain barrier and neurons [79]. In conclusion, although the studies concerning the role played by NCX in the pathological mechanisms of brain injury during neurodegenerative diseases have had a late beginning compared with those concerning heart disease, the availability of pharmacological agents able to selectively modulate each NCX subtype activity and antiporter mode of operation will enable researchers to gain fundamental insights into its pathophysiological role and, thus, to obtain more tangible perspectives on how to treat such neurological disorders.

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The ‘‘Loop’’ Diuretic Drug Bumetanide-Sensitive Naþ-Kþ-Cl– Cotransporter in Cerebral Ischemia Dandan Sun

1 Introduction The Naþ-Kþ-Cl– cotransporter (NKCC) belongs to the cation-chloride cotransporter superfamily (CCC), which mediates the coupled movement of Naþ and /or Kþ with Cl– across the plasma membrane [1–3]. Two isoforms of NKCC (NKCC1 and NKCC2) have been identified as two different gene products, human genes SLC12A1 (NKCC2) and SLC12A2 (NKCC1) [1,4]. Together with the thiazide-sensitive renal Naþ-Cl– cotransporter (NCC; SLC12A3), they constitute the Naþ-dependent branch of the CCC superfamily. NKCC1 and NKCC2 share 50–60% amino acid sequence homology and have distinct tissue distributions. NKCC1, which was first identified on the basolateral membranes of secretory epithelial cells, is referred to as the ‘‘secretory’’ or ‘‘house-keeping’’ NKCC [1,4]. In addition to secretory epithelial cells, NKCC1 is expressed in a wide variety of tissues including epithelial cells and nonepithelial cells, such as neurons and glia [2,5]. NKCC1 plays a role in salt secretion and absorption, cell volume regulation, and maintenance of intracellular Cl– concentration ([Cl–]i) [1,2,5]. NKCC1-null mice exhibit multiple tissue phenotype changes and functional disorders, including deafness, decreased blood pressure, intestinal bleeding, infertility, and salivary secretion reduction [5–7]. These findings imply that NKCC1 is important in cellular function in a wide variety of tissues. In contrast to NKCC1, NKCC2 expression is confined to the apical membrane of the epithelial cells of the thick ascending limb of Henle’s loop in kidney [1]. Thus, NKCC2 is important in regulation of extracellular fluid volume and osmolarity. NKCC2 mutation is associated with Bartter syndrome, a renal tubular disorder [5]. Loss of ionic homeostasis plays a central role in pathogenesis of ischemic cell damage. During ischemia, intracellular Kþ decreases, while intracellular Ca2+, D. Sun (*) Department of Neurological Surgery, University of Wisconsin School of Medicine and Public Health, Madison, WI, 53705, USA e-mail: [email protected]


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Naþ, Cl–, along with H2O increase [8]. Ischemia-induced perturbation of ionic homeostasis leads to subsequent activation of proteases, phospholipases, and formation of oxygen and nitrogen free radicals. This cascade of signal transduction events results in long-term functional and structural changes in membrane and cytoskeletal integrity and eventual cell death. Results from in vitro and in vivo studies demonstrate a role for NKCC1 in ischemia-induced dissipation of ionic homeostasis. Therefore, the ion transport protein could provide a novel target for the future development of therapeutic treatment of ischemic cerebral damage. This chapter focuses on the role of NKCC1 in the central nervous system (CNS), especially under ischemic pathophysiological conditions. Interested readers are referred to a series of reviews that cover broader aspects of NKCC and Kþ-dependent chloride transporters [2,5,9].

2 NKCC1 Gene Structure and Structural Features Using cDNA probes, a NKCC1 human homolog has been identified in human colonic carcinoma line T84 cell [10]. Analysis of human NKCC1 (hNKCC1) gene SLC12A2 (3,498 kb) found that hNKCC1 is located in humans at chromosome 5q23 and composed of 1212 amino acids with a molecular mass of 132 kDa. The primary structure of hNKCC1 is 74% identical to the sequence of shark rectal gland Na-K-Cl cotransporter (sNKCC1) and 91% identical to a mouse Na-K-Cl cotransporter (mNKCC1) [10]. The molecular structure of NKCC1 has been proposed based on cDNA sequence analysis. The sequence analysis of sNKCC1 reveals that NKCC1 protein has 12 putative transmembrane domains (TM), flanked by large cytoplasmic amino and carboxyl termini, which contain regulatory phosphorylation sites [4,1] (Fig. 1). There are two potential N-linked glycosylation sites predicted on an extracellular loop between putative transmembrane segments 7 and 8 [4]. NKCC1 in different tissues have molecular weights that vary from 120 to 190 kDa [2]. They form homodimers in plasma membrane via COOH-terminus [11]. The proposed topology is supported by the recent experimental data using an in vitro translation system [12].

3 Molecular Biology Site-directed mutagenesis studies show that changes in Naþ and Rbþ (Kþ) affinity result from changes in TM2 whereas Cl– affinity was affected by changes in TM4–7 [13]. The affinity for bumetanide, a potent inhibitor of NKCC, was found to be affected by residues in the TM2–7 region, and also by residues in TM11 and TM12 [13]. In contrast, the conserved hydrophilic C-termini of hNKCC1 and sNKCC1 are not involved in the difference in affinities for the transported ions [14]. Therefore, the sequences within TM2 segments affect

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Fig. 1 Model of Naþ-Kþ-Cl– cotransporter protein based on its hydropathy profile. The NKCC protein is comprised 12 transmembrane (TM)-spanning domains, numbered TM1TM12. Glycosylation sites are predicted on an extracellular loop between putative transmembrane segments 7 and 8. Identified phosphorylated threonine residues are indicated by ‘‘*’’ with an adjacent ‘‘p’’ (produced by the kind permission of Curr. Opin. Nephrol. Hypertens from [1])

transport affinity for cations, but not for Cl–. The bumetanide binding probably requires conformational interaction between TM and extracellular domains.

4 Tissue and Cellular Distribution Tissue distribution analysis by Northern blot revealed presence of NKCC1 transcripts in all tissues [9]. Because of NKCC1’s location in basolateral membrane of epithelia, NKCC1 is important in transcellular ion transport in secretory epithelia. In the CNS, NKCC1 is expressed in neurons, glia, choroid plexus, and cerebral vascular endothelial cells [3,9]. In addition to salt transport, NKCC1 is also involved in maintenance and regulation of cell volume in both epithelial and nonepithelial cells. Moreover, NKCC1 functions in accumulation of intracellular Cl– and regulation of Cl– homeostasis in the CNS. Therefore, NKCC1 is important in GABAergic signaling and changes of neuronal excitability [15]. The role of NKCC1 in epileptic formation has been reviewed recently [16].

5 Biophysical and Electrophysiological Properties There are three fundamental characteristics of NKCC function [2]. First, ion translocation by NKCC requires that all three ions (Naþ, Kþ, Cl–) are simultaneously present on the same side of the membrane [2]. Second, ion transport mediated by NKCC is electroneutral in most cases, with a stoichiometry of 1

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Naþ:1Kþ:2 Cl– [2]. The driving force for ion influx is supplied by the combined chemical gradients for three transported ions [2], with a net electrochemical potential.

ð

Dm ¼ RT ln

½Naþ o ½Kþ o ½Cl 2o ½Naþ i ½Kþ i ½Cl 2i

Þ

:

Third, NKCCs are targets of the ‘‘loop’’ diuretics. Sulfamoybenzoic acid loop diuretics such as bumetanide bind to NKCCs and can inhibit ion translocation of three ions [17]. Bumetanide (IC50 0.3 mm) is the most widely used drug in NKCC1 inhibition and more potent than other sulfamoybenzoic acid loop diuretics such as furosemide (IC50 7 mm) [18].

6 Regulatory Mechanisms – Regulation of NKCC1 via Phosphorylation NKCC1 activity is regulated by multiple stimuli such as a decrease in [Cl–]i, hypertonic stress, an increase in [Ca2+]i, and a-adrenergic receptor stimulation [19–22]. Cl–-mediated regulation of NKCC1 activity has been extensively studied in epithelial cells. Epithelium secrets Cl– through Cl– channels located on the apical membrane. A decrease in [Cl–]i will increase the activity of basolateral membrane NKCC1, which will bring Cl– into the cell, thereby promoting continuous Cl– secretion across the apical membrane [2]. Lytle et al. have demonstrated that a decrease in [Cl]i stimulates both NKCC1 activity and NKCC1 protein phosphorylation in epithelial cells [23,24]. The degree of NKCC1 activation is closely related to its phosphorylation and this suggests that the Cl–-mediated regulation of NKCC1 activity is via phosphorylation [24]. Sequence analysis has revealed that Thr184, Thr189, Thr202, and Thr1114 of N- and C-termini are the conserved regulatory phosphoracceptor residues in NKCC1 [4,19]. Both kinases and phosphatases contribute to the precise regulation of NKCC1 activity through their opposite effects on NKCC1. Recently, several kinases have been discovered to mediate NKCC1 phosphorylation. OSR1 (oxidative stress response kinase) and SPAK (STE20/SPS-1 related, proline–alanine-rich kinase), along with its rat homolog PASK (proline–alanine-rich STE20-related kinase), were found to bind to the third conserved domain in the NKCC1 N-terminus and co-immunoprecipitated with NKCC1 protein [25,26]. Overexpression of PASK in HEK cells causes a small but significant increase in both hNKCC1 and sNKCC1 activity and the phosphorylation of PASK coincides with that of NKCC1 [25]. PASK and SPAK are preferentially expressed in the brain and in cells that are active in ion transport and rich in Naþ/Kþ-ATPase [26–28]. PASK is suggested as a stress-related kinase due to its putative caspase cleavage site and


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its ability to activate p38 MAPK pathway [28]. Neurons, glia, and epithelial choroid cells in the CNS have shown PASK expression [29]. On the other hand, protein phosphatase is responsible for dephosphorylation of NKCC1. The catalytic subunit of protein phosphatase 1 binds to the proteins that contain the consensus motif RVXFXD [30]. The N-terminus of NKCC1 contains a highly conserved RVNFVD sequence both in sNKCC1 (107–112) and hNKCC1 (140–145) [30]. The RVXFVD motif mutation produces an activation shift in NKCC1 regulation by Cl–. Dowd et al. [25] have shown that dominant negative PASK (DNPASK) decreases the activity of hNKCC1 by 80% and sNKCC1 by 60%. In addition, protein phosphatase type 1 inhibitor calyculin A nearly completely restores NKCC1 activity in the DNPASK transfected cells [25]. These findings strongly suggest that an alteration in phosphorylation/ dephosphorylation of NKCC1 regulates NKCC1 activity. Recent studies have demonstrated that a novel WNK family of serine–threonine kinases (kinases with no K = lysine, which is substituted with cysteine) plays an important role in the regulation of epithelial Naþ channels and ion transporters including NKCC1 [16]. In humans, there are four genes encoding WNK1, WNK2, WNK3, and WNK 4, respectively. WNK3 is most highly expressed in brain. Co-expression of WNK3 and NKCC1 results in an increase in NKCC1 activity and induces robust phosphorylation of threonine212 and threonine217, the known regulatory sites in NKCC1 [31]. Moreover, it has been suggested that WNK1/WNK4 can phosphorylate SPAK/OSR1, which in turn directly phosphorylates NKCC1 [16]. Therefore, NKCC1 function can be dynamically modulated by changing activities of the kinases and phosphatases.

7 Physiological Properties – NKCC1-Mediated Regulation of Kþ Uptake, Intracellular Cl–, and Cell Volume in the CNS NKCC1 is expressed in neurons, glia (astrocytes and oligodendrocytes), as well as choroid plexus epithelial cells and blood vessel endothelial cells [32–35]. Functional studies demonstrate that NKCC1 plays an important role in regulation of [Cl–]i, Kþ uptake, and cell volume of cells in the CNS [15,36]. g-Aminobutyric acid (GABA) and glycine are the main inhibitory neurotransmitters in the adult mammalian CNS. Activation of GABA receptors results in Cl– influx and hyperpolarization of the plasma membrane. However, during fetal and postnatal development, the equilibrium potential for Cl– is more positive than the resting potential and activation of GABA receptors triggers an excitatory response and a Cl– efflux [37–39]. The excitatory action of GABA in the immature CNS is important for the development of the nervous system [40]. Cells maintain Cl– homeostasis through the fine balance between inwardly directed Cl– flux and Cl–extrusion systems. NKCC1 functions as a Cl– influx system and contributes to the active accumulation of intracellular Cl– in

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immature neurons [15,39]. An emerging hypothesis asserts that the balance between the inwardly directed NKCC1 and the outwardly directed neuronal K-Cl cotransporter (KCC2) may be important in the determination of GABAA receptor-mediated depolarizing responses. This view is supported by the findings that expression of the inwardly directed NKCC1 in neurons precedes expression of the outwardly directed KCC2 [41]. Additionally, genetic ablation of NKCC1 leads to a decrease in resting [Cl–]i and abolishes the GABAmediated depolarization in mouse dorsal root ganglion cells [15]. Disruption of KCC2 results in frequent seizures, abolishment of respiration, and early lethality in mice due to anomalous excitatory actions of GABA and glycine [42]. Opening of the GABA-activated Cl– channel or depletion of intracellular Cl– significantly stimulates NKCC1 activity in neurons [21]. The GABA-mediated loss of intracellular Cl–, but not a subsequent membrane depolarization or shrinkage, leads to stimulation of NKCC1 [22]. This stimulation might be an important positive feedback mechanism to maintain intracellular Cl– level and GABA function in immature neurons. The depolarizing effect of GABA is of functional importance during neuronal maturation and differentiation. The depolarizing action of GABA subsequently activates NMDA and voltage-gated Ca2+ channels that leads to a rise in intracellular Ca2+ and activation of a wide range of intracellular signaling pathways. Importantly, synergistic excitatory actions of GABAA and glutamergic NMDA receptor have been found in the neonatal hippocampus [40]. The interplay between GABA and glutamate-mediated excitatory actions is required for the induction of synaptic plasticity during the development [40,43]. Interestingly, NKCC1 in immature cortical neurons is stimulated by group-I mGluR- and glutamate inotropic receptors NMDA- and AMPA-mediated signal transduction pathways [21,22]. The effects are dependent on a rise of intracellular Ca2+. A synergistic interaction between GABAA and NMDA receptors has been reported in formation of synchronous Caþ2 oscillations [40]. Stimulation of NKCC1 by glutamate could increase intracellular Cl– that would reinforce the depolarizing actions of GABA [21]. In addition to neurons, GABA also triggers a depolarizing efflux of Cl– in cultured oligodendrocytes [44]. Re-accumulation of intracellular Cl– after depletion is blocked by removal of extracellular Naþ or inhibition of NKCC1 activity [45]. Thus, NKCC1 may be important in maintaining high [Cl–]i in oligodendrocytes. While the trophic effect of GABA on neurons is now well established [46,47], the effect of GABA on oligodendrocyte survival and growth remains uncertain. NKCC1 is present and active in rat spinal cord oligodendrocytes [35]. Expression of NKCC1 protein in the rat spinal cord is increased during development. In cultured oligodendrocytes, 39% of the total Kþ (86Rbþ) influx represents NKCC1 activity. Activation of GABAA receptors with muscimol produces a reduction in intracellular Cl– content and a stimulation of NKCC1 activity. Muscimol also triggers an increase in [Ca2+]i; this increase depends on NKCC1 activity. Survival of oligodendrocytes following withdrawal of growth factors is enhanced by muscimol; this effect also requires


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NKCC1 activity [35]). These results imply that NKCC1 functions in rat oligodendrocytes to maintain [Cl–]i above electrochemical equilibrium, and that it is required for GABAergic trophic effects. Both astrocytes and the choroid plexus epithelia have been suggested to play an important role in Kþ uptake in the CNS. Co-localization of NKCC1 and glial fibrillary acidic protein (astrocyte marker protein) was detected in astrocytes of cortex, corpus callosum, hippocampus, and cerebellum of rat [48]. Expression of NKCC1 was also observed in the perivascular astrocytes of cortical cortex, cerebral white matter, and hippocampus [48]. One of the major functions of astrocytes in the CNS is to buffer the extracellular Kþ concentration by transporting the Kþ released by active neurons [49]. Astrocytes take up Kþ, which can then leave astrocytes at remote locations where extracellular Kþ concentrations have not risen, or Kþ can efflux at the endfeet of the astrocyte where it is subsequently transported by blood vessels [49]. NKCC1 in the processes of the perivascular astrocytes is co-localized with AQP4, the latter is abundantly expressed in the astrocyte endfeet [50]. Therefore, these data suggest that NKCC1 may play a role in transport of Kþ from brain to blood to restore the extracellular Kþ concentration after neuron firing cessation. Lastly, the epithelial cells in choroid plexus are important for cerebrospinal fluid production. NKCC1 is constitutively active in choroid plexus epithelial cells and functions to reabsorb Kþ from the CSF, therefore, buffering and regulating brain interstitial Kþ homeostasis [33]. In addition to Kþ uptake, NKCC1 may also function in the regulation of intracellular Cl- in astrocytes. In acutely isolated hippocampal astrocytes from mature Sprague-Dawley rat, intracellular Cl- level is higher than it is predicted by passive distribution and the Cl--equilibrium potential is far more positive than the membrane potential [51]. Application of GABA in the CA3 region of the neuronfree rat hippocampal slice induced depolarization of astrocytes [52]. A similar GABAA receptor-induced depolarization has also been observed in acutely isolated astrocytes from adult Sprague-Dawley rat or cultured mature hippocampal astrocytes from neonatal Wistar rat [51,53]. These findings suggest that NKCC1 may play a role in maintaining high intracellular Cl- levels in astrocytes. NKCC1 activity is important in maintaining cell volume. Inhibition of NKCC1 activity or genetic ablation of NKCC1 impairs regulatory volume increase in astrocytes and neurons following hypertonicity-induced cell shrinkage [22,52])

8 Pathophysiological Relevance in Stroke 8.1 NKCC1 Contributes to Neuronal Damage During Acute Neurotoxicity Cl movement has been shown to be a central component of the acute excitotoxic response in neurons [54,55]. The acute excitotoxicity is thought to be

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mediated by excessive depolarization of the postsynaptic membrane. Depolarization results in an osmotic imbalance, which is countered by an influx of Cl-, Naþ, and water and leads to cell lysis. Removal or reduction of Cl– from the extracellular medium during excitatory amino acid (EAA) exposure completely eliminates the acute excitotoxic response in hippocampal [54] and retinal neurons [55]. NKCC1 is important in maintenance of [Cl]i in neurons as described above [15,37,39,56]. Thus, NKCC1 may affect neuronal excitability through regulation of [Cl-]i [15,57]. Activation of the ionotropic glutamate NMDA receptor, the AMPA receptor, and the metabotropic glutamate receptor (group-I) significantly stimulates NKCC1 activity in SH-SY5Y neuroblastoma cells [56]. Interestingly, inhibition of NKCC1 activity with its potent inhibitor bumetanide abolishes glutamate-mediated neurotoxicity and significantly attenuates oxygen and glucose deprivation (OGD)-induced neuronal death (Fig. 2, [58]). Furthermore, NMDA-mediated increases in 36Cl content and intracellular Naþ overload are reduced in the presence of bumetanide [58]. Bumetanide also blocks NMDA-induced neuronal swelling [58]. Thus, NKCC1 may act as another mechanism that contributes to Naþ and Cl– overload during glutamate-mediated acute excitotoxicity. This view is further supported by the

Fig. 2 Inhibition of NKCC1 decreases glutamate-mediated neurotoxicity. Cell mortality in cultured neurons was assessed by propidium iodide and calcein staining after 24-h treatment with glutamate or NMDA. A. Data are means SE. * P < 0.05 vs. control, ** P < 0.05 vs. NMDA, # P < 0.05 vs. glutamate. B. Bumetanide was added 30 min prior to the glutamate treatment and cells were assessed at 24 h post-treatment. * P < 0.05 vs. control, # P < 0.05 vs. glutamate (adapted from [58])


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similar levels of neuroprotection exhibited in neurons from NKCC1-null (NKCC1–/–) mice [3]. In addition, in neurons with injured axons, Cl– accumulation is attributable to NKCC1-mediated Cl– influx and a decrease in Cl– extrusion by KCC2 [59]. Cl– accumulation causes a switch in GABA action from inhibitory to excitatory and leads to excitotoxicity [59]. Blockage of Naþ and Cl– entry by removal of extracellular Naþ and Cl– abolishes excitotoxic dendritic injury and NMDA-mediated neurodegeneration in retinal ganglion cells [60,61]. Downregulation of KCC2 and a subsequent decrease in GABAmediated inhibition may contribute to excitotoxicity in injured neurons [62]. These results imply that stimulation of NKCC1 activity and downregulation of KCC2 are important in ischemic neuronal damage.

8.2 NKCC1-mediated Ionic Dysregulation, Swelling, and Excitatory Amino Acid Release in Astrocytes Following Ischemia Glial cells outnumber neurons by 10 times and occupy up to 50% of the human brain volume [63]. Glial cells have a critical function in the glutamate–glutamine shuttle, lactic acid supply for neuronal energy metabolism, regulation of synaptic and perisynaptic glutamate levels, regulation of ion concentrations (Hþ, Kþ, and Ca2+) in the intracellular and extracellular spaces, and the coupling of neuronal activity and cerebral blood flow [64, 65]. Therefore, alterations of astrocyte function after ischemia could have a significant impact on cerebral brain damage. Early loss of astrocytes is a principal feature of hyperglycemic and complete ischemia [66]. Despite the importance of astrocytes in neurodegeneration, the mechanisms underlying ischemic astrocyte damage are poorly understood. The following research findings illustrate the role of NKCC1 in ischemia-induced astrocyte damage. 8.2.1 High [Kþ]o-mediated Astrocyte Swelling Astrocytes undergo rapid swelling in a number of acute pathological states, such as ischemia and traumatic brain injury [67]. Unresolved astrocyte swelling will have detrimental effects such as reduction of extracellular space, a decrease of normal cerebral blood flow, and an accumulation of EAAs such as glutamate [68]. In addition, swollen astrocytes would have a diminished capacity to perform their normal homeostatic functions. Considerable work has been focused on the role of cation-chloride cotransporters in maintenance and regulation of astrocyte volume. The expression of NKCC1 protein is found in astrocytes [48], which could function in clearing of excessive [Kþ]o as mentioned above [69]. During ischemia, one significant pathophysiological change in the CNS is an

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elevation of [Kþ]o. A few minutes of anoxia/ischemia raises [Kþ]o to 60 mM [8]. NKCC1 in astrocytes may play a role in Kþ uptake under high [Kþ]o conditions. In cultured astrocytes, 75 mM [Kþ]o causes NKCC1-mediated Kþ influx to be stimulated by 80% [70]. The high [Kþ]o-induced activation of NKCC1 is completely abolished by either removal of extracellular Ca2+ or by blocking the L-type voltage-dependent Ca2+ channels with nifedipine [71]. Additionally, intracellular 36Cl accumulation increases significantly in response to 75 mM [Kþ]o and is abolished by inhibition of NKCC1 activity either by bumetanide or genetic ablation [72,71]. These data suggest that the cotransporter activity is stimulated under high [Kþ]o via Ca2+-mediated signal transduction pathways. Seventy-five millimolar [Kþ]o also triggers cell swelling in NKCC1þ/þ astrocytes by 20% [70]. This high [Kþ]o-mediated swelling is abolished by inhibition of NKCC1 activity, either with bumetanide or genetic ablation [72,70]. In addition, high [Kþ]o-induced astrocyte swelling is also observed in the rat optic nerve model [73]. Furosemide and bumetanide reversibly suppress the high [Kþ]o-induced astrocyte swelling in enucleated nerves [73]. Additionally, astrocytes in optic nerves exhibited a Ca2+-independent swelling during ischemia that was blocked by bumetanide [24]. Taken together, these studies suggest that NKCC1 activation leads to astrocyte swelling. 8.2.2 High [Kþ]o-Mediated Excitatory Amino Acid Release from Astrocytes Glutamate is the principal excitatory neurotransmitter in the mammalian CNS. Elevation of glutamate in the extracellular space and excessive activation of NMDA receptors cause cell death [75]. Astrocytes maintain a large transmembrane glutamate gradient, with [Glu]i of 2–10 mM whereas [Glu]o is approximately 1 mM [76]. This gradient results from the high density of Naþ-dependent glutamate transporters in astrocytes, which regulate synaptic transmission by controlling glutamate diffusion and concentration in the extracellular space [77]. Therefore, reduced glutamate uptake or release of sequestered glutamate from astrocytes may contribute to excitotoxicity under pathophysiological conditions [67]. For example, one consequence of high [Kþ]o-induced astrocyte swelling will be the release of glutamate via activation of volume-sensitive organic anion channels (VSOACs) [71,78]. VSOACs are ubiquitously expressed chloride channels, which are permeable to a variety of small organic anions, including the amino acids taurine, glutamate, and aspartate [79]. In response to swelling, VSOAC-mediated efflux of amino acids can function as a regulatory volume decrease mechanism in astrocytes [82]. Release of [14C]-D-aspartate from astrocytes, which serves as a surrogate for glutamate, is negligible under control conditions [70,80]. However, high [Kþ]o triggers a significant release of [14C]-D-aspartate from astrocytes. High [Kþ]o-induced release of [14C]-Daspartate is abolished by the anion channel blocker DIDS, suggesting an involvement of VSOACs [53,69]. When NKCC1 is inhibited by bumetanide or genetic ablation, this high [Kþ]o-induced aspartate release is reduced by


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30–50% (Fig. 3) [72,70]. On the other hand, stimulation of NKCC1 under high [Kþ]o conditions leads to increases in intracellular Naþ, which could slow or even reverse Naþ-dependent glutamate transporters and increase glutamate release from astrocytes. Taken together, astrocyte NKCC1 stimulation will contribute to excitotoxic injury by increasing swellinginduced glutamate release or decreasing glutamate clearance from the extracellular space.

Fig. 3 Effect of bumetanide on the high-[Kþ]o-mediated release of preloaded D-[14C]aspartate from astrocytes. A. Cells loaded with D-[14C]aspartate were perfused with 75 mM [Kþ]o for 20 min. B. Astrocytes were exposed to bumetanide for 20 min and then exposed to 75 mM [Kþ]o + bumetanide. Cells were then washed with normal buffer and re-exposed to 75 mM [Kþ]o. (adapted from [72,71])

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8.2.3 Naþ and Cl– Dysregulation in Astrocytes Following Oxygen and Glucose Deprivation Loss of the trans-plasma membrane Naþ gradient is one of the key elements in promoting cellular damage in astrocytes during ischemia [81]. An increase in intracellular Naþ is found in rat spinal cord astrocytes [82], rat cortical astrocytes [81], and mouse cortical astrocytes [83] under ischemic conditions. While decreased activity of Naþ/Kþ-ATPase during ischemia will lead to reduced Naþ extrusion, the activation of several Naþ entry pathways, including NKCC1, also contributes to ischemia-induced loss of the Naþ homeostasis. OGD in astrocytes results in both an increase in NKCC1 phosphorylation and 86Rb influx [84]. This stimulation of NKCC1 activity is accompanied by an accumulation of 36Cl and 3.5-fold increase in [Naþ]i in astrocytes [85]. Either bumetanide or genetic ablation of NKCC1 significantly reduces the OGD/REOXmediated increase in [Naþ]i and [Cl–]i. Given that the extracellular ionic composition during hypoxia/ischemia is featured with a lower concentration of Naþ, Cl–, Ca2+ and a higher concentration of Kþ as well as Hþ [86], it is important to examine whether NKCC1 still functions in this altered extracellular ionic environment. Thus, an alternative in vitro hypoxic model was established by perfusing cells with hypoxic, acidic, ion shifted-Ringers (HAIR) that mimics the extracellular ionic and oxygen composition in vivo. Following 5 min hypoxia, [Naþ]i raises rapidly over the first 5 min REOX and plateaus to a value approximately four-fold of control and is sustained over the following 45 min REOX. Bumetanide or genetic ablation of NKCC1 reduces hypoxia-mediated rise in [Naþ]i by 65% [87]. The HAIRmediated increases in [Naþ]i are doubled when Naþ/Kþ-ATPase activity is blocked with ouabain [87]. This suggests that the NKCC1 mediates a Naþ overload that cannot be offset by Naþ/Kþ-ATPase activity. 8.2.4 Ca2+ Dysregulation in Astrocytes Following Oxygen and Glucose Deprivation NKCC1-mediated increases in [Naþ]i following in vitro ischemia have been linked to Ca2+ loading and subsequent mitochondrial damage. As a consequence of increases in [Naþ]i, Naþ/Ca2+ exchanger, (NCX)-mediated Ca2+ extrusion is reduced or NCX may function in a reverse mode (NCXrev), which leads to increases in [Caþ2]i (Fig. 4). Thus, NKCC1 and NCXrev may contribute to intracellular Naþ and Caþ2 overload in astrocytes following in vitro ischemia [85]. In addition, Naþ and Caþ2 overload in ischemic astrocytes leads to a dissipation of the mitochondrial membrane potential and release of mitochondrial cytochrome c, which can be attenuated by bumetanide or with genetic ablation of NKCC1 [87]. These findings suggest that the concerted activities of multiple ion transport proteins are important in the perturbations of Naþ and Ca2+ homeostasis and in astrocyte damage in response to hypoxia/ischemia.


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Fig. 4 Increase in [Ca2þ]i following HAIR/REOX depends on NKCC1 and NCX activity. A. [Ca2+]i was monitored in NKCC1þ/ + or NKCC1–/– astrocytes during 15 min HAIR and 45 min REOX. In studies involving KB-R7943 an inhibitor of NCXrev, the drug was present 30 min before HAIR and during REOX. B. Summary data of changes in [Ca2+]i at 45 min REOX. Data are means SE. * P < 0.05 vs. control, # P < 0.05 vs. NKCC1þ/+ HAIR/REOX. (adapted from [87])

8.3 NKCC1 in AMPA-Mediated Oligodendrocyte Excitotoxicity The primary role of oligodendrocytes in the CNS is the production of myelin, which forms an insulating sheath around the axons of neurons and allows for efficient electrical transmission [88]. Loss of oligodendrocyte function is involved in a number of neurodegeneration diseases. Likewise, early ischemic damage to white matter involves oligodendrocyte and axon damage [89]. NKCC1 plays a role in oligodendrocyte damage following activation of nonNMDA glutamate receptors. AMPA/kainate receptor-mediated excitotoxcity results in spinal cord white matter injury in a process that is linked to oligodendrocyte damage and axonal demyelination [90]. Indeed, the vulnerability of immature oligodendrocytes to ischemic injury is a major component of the brain injury associated with cerebral palsy [91]. Oligodendrocytes are thought to express primarily non-NMDA type ionotropic glutamate receptors, although recent studies suggest that NMDA-type glutamate receptors may predominate in processes, while AMPA/kainate receptors predominate on the soma [92]. Exposing cultured oligodendrocytes to AMPA plus the AMPA desensitization blocker cyclothiazide (CTZ) leads to a transient rise in [Ca2+]i, which is followed by a sustained intracellular [Ca2+]i overload, NKCC1 phosphorylation, and a NKCC1-mediated Naþ influx [93]. In the presence of a specific AMPA receptor inhibitor 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX), AMPA/CTZ fails to elicit any changes in [Ca2+]i. The AMPA/CTZ-induced sustained [Ca2+]i rise led to mitochondrial Caþ2 accumulation, release of cytochrome c from mitochondria, and cell death [93]. The AMPA/CTZ-elicited [Ca2+]i increase, mitochondrial damage, and cell death are significantly reduced by inhibiting NKCC1 or NCXrev. These data show that in cultured

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oligodendrocytes, activation of AMPA receptors leads to NKCC1 phosphorylation that enhances NKCC1-mediated Naþ influx. The latter triggers NCXrev-mediated [Ca2+]i overload and compromises mitochondrial function and cellular viability [93]. Such a link between Naþ overload and activation of NCXrev has been observed under other conditions [94]. NCXrev causes Ca2+ influx after NMDA and non-NMDA receptor activation in depolarized and glucose-deprived neurons [95,96]. Increase in intracellular Naþ and the subsequent induction of NCXrev have also been found in mechanical strain injury of astrocytes [97] and in axonal damage [94].

8.4 NKCC1 in Ischemic Hippocampal Damage Several studies have indicated that OGD in the brain slice induces an accumulation of [Cl–]i in area CA1 hippocampal neurons [98–101]. [Cl–]i started to increase during OGD and accumulated throughout REOX, reaching levels twice as high as those found immediately after OGD [98]. In another study, Cl– content in the cytoplasm, mitochondria, and nuclei was increased by 50% after 10 min OGD and by 300% at 30 min REOX following 10 min OGD [99]. The sustained rise in [Cl–]i following OGD can directly induce neuronal damage [100]. Given the apparent significance of the OGD-mediated rise in [Cl–]i, considerable effort has focused on the underlying mechanisms. Recent studies indicate that the rise in [Cl–]i during OGD is likely to be due a number of different Cl– transport mechanisms, including Cl– transporters and Cl– channels [101]. In contrast, the rise in [Cl–]i during REOX can be completely prevented by bumetanide [101]. Increased phosphorylation of NKCC1 has been demonstrated after OGD, consistent with the timing of changes of [Cl–]i [101]. NKCC1 activity may be primarily responsible for the changes in [Cl–]i during REOX.

8.5 NKCC1 in In Vivo Cerebral Ischemia While in vitro studies are useful for demonstrating proof of principle for a role of NKCC1 in cerebral ischemia, it is important to extend these studies by using in vivo animal models. The most widely used model of focal cerebral ischemia is the endovascular suture occlusion of the middle cerebral artery (MCAO) in rodents. Expression of NKCC1 detected with T4 antibody immunoreactive signals is observed in cell bodies and dendrites of pyramidal neurons in cerebral cortex of sham rats. Two hours of focal cerebral ischemia and 24 h reperfusion lead to increased NKCC1 immunostaining in neurons scattered in the ischemic cortex and striatum [34]. Immunoblotting revealed that expression of NKCC1 protein was increased following 2 h focal ischemia in cerebral cortex and striatum. A sustained upregulation of NKCC1 in cortex was detected at 4, 8,


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12, and 24 h of reperfusion [34]. An increase in the phosphorylated NKCC1 (NKCC1-p) was also found at 4 and 8 h of reperfusion in the ipsilateral cortex [34]. Because the change in phosphorylation state of NKCC1 is a major regulatory mechanism that stimulates NKCC1 activity, these findings suggest that elevated NKCC1 expression and activity may be involved in cerebral ischemic damage. Upregulation of NKCC1 is found in hippocampus in gerbils after 5 min global ischemia and 24 h reperfusion [102]. However, in the same model there is also a transient downregulation of NKCC1 protein expression at 12 h reperfusion [102], which may be a compensatory reaction to protect neurons from excitotoxicity. Two hours of MCAO and 24 h reperfusion result in an average infarction volume of 238.3 mm3 in spontaneous hypertensive rats [34]. However, when 100 mM bumetanide is continuously infused via microdialysis into the cortex during MCAO and reperfusion, the resulting infarct was reduced by 25%. Edema induced by MCAO was also reduced by 79% in the bumetanide-treated brains [34]. This indicates that NKCC1 contributes to cerebral ischemic cell damage and edema in vivo. In rats subjected to permanent MCAO, intravenous administration of bumetanide (7.6–30.4 mg/kg) immediately before occlusion attenuates edema formation as determined by magnetic resonance imaging [103]. This further suggests a role for NKCC1 in the edema formation during cerebral ischemia. In addition to pharmacological blockage of NKCC1, genetic ablation of NKCC1 can be used to further establish that NKCC1 contributes to ischemic damage. Two hours MCAO and 10 or 24 h reperfusion caused infarction (85 mm3) in NKCC1 wild-type (NKCC1þ/+) mice (Fig. 5). Infarction volume in NKCC1–/– mice was reduced by 30%–46% [3]. Heterozygous mutant (NKCC1þ/–) mice also showed 28% reduction in infarction [3]. Brain edema was significantly increased after 2 h MCAO and 24 h reperfusion in NKCC1þ/+ mice. In contrast, there was 50% less edema formation in either NKCC1þ/– or NKCC1–/– mice [3]. These results are consistent with the neuroprotective effects in rats mediated by pharmacological inhibition of NKCC1 with bumetanide. White matter ion homeostasis, especially Naþ and Kþ, is crucial to normal axonal functions and dissipation of ionic homeostasis will lead to irreversible injury. Amyloid precursor protein (APP) is transported by fast anterograde axon transport along microtubules and has recently been shown as a sensitive marker of axonal disruption in white matter during brain ischemia [104]. Disruption of this transport as a consequence of cytoskeleton derangement results in APP accumulation, indicating the dysfunction or discontinuity of the axons [105]. Two to 4 h focal ischemia causes cytoskeletal breakdown and disturbance of fast axonal transport in myelinated fiber tracts [105]. NKCC1þ/+ brains exhibit significant accumulation of APP in the ipsilateral internal capsule after 2 h focal ischemia or 10 h reperfusion. In contrast, APP accumulation is reduced by 55% in NKCC1–/– brains after 2 h focal ischemia and 10 h reperfusion [3]. White matter degeneration

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Fig. 5 Brain infarction is decreased in NKCC1–/– mice. A. Coronal sections of brains from NKCC1þ/+- or NKCC1–/– mice after 2 h MCAO and 10 or 24 h reperfusion were stained with TCC and infarct volume was calculated. B. Summarized infarct volume. Data are means SD, P < 0.05 vs. NKCC1þ/ + . Scale bar 5 mm. (adapted from [3])

is often assumed to be secondary to neuronal damage. However, no changes in myelin basic protein expression were observed after 2 h MCAO and 24 h reperfusion [3]. This implies that APP accumulation occurs early and is likely a result of primary axonal ischemic damage. Taken together, these results suggest that NKCC1 plays a role not only in gray matter but also in white matter ischemic damage. The in vitro studies in astrocytes or in oligodendrocytes show that NKCC1 activation leads to Naþ overload, which triggers NCXrev after ischemia and excitotoxicity [85,87,93]. Although these findings support that NKCC1 and NCXrev may collectively contribute to ischemic cell damage, it has not yet been tested in in vivo ischemic models until recently. Luo et al [106] examined whether reduction of NKCC1 and NCX1 could provide neuroprotection after in vitro or in vivo ischemia. Both neurons and astrocytes cultured from double heterozygous (NKCC1þ/–/NCX1þ/–) mice showed considerably reduced expression and function of NKCC1 and NCX1 [106]. In addition, as compared to NCX1þ/+ mice, infarct volume at either 24 or 72 h reperfusion following 30 min MCAO is reduced by 50% in NKCC1þ/–/NCX1þ/– mice [106]. Taken together, these studies demonstrate that NKCC1 plays a role in ischemic brain damage.


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9 Therapeutic Perspectives NKCC1 has a broad tissue distribution and serves to maintain cellular ion homeostasis. Over the past decades the function and regulation of NKCC1 have been extensively focused in epithelial cells. The study of the role of NKCC1 in the CNS is relatively new and exciting. NKCC1 protein is expressed in multiple cell types throughout the brain. Through the regulation of intracellular Cl–, NKCC1 activity is able to affect GABAergic signaling and neuronal excitability. Therefore, NKCC1 is being explored as a target for anti-convulsant therapy in neonates [107]. Preclinical in vitro and in vivo experimental studies also demonstrate that NKCC1 activation contributes to ischemic neuronal damage. Thus, bumetanide could be useful for reducing ischemic damage, edema, and seizures that are secondary to ischemic brain injury. Controls of bumetanide’s action in the periphery system to limit systemic side effects in kidney and electrolyte homeostasis should be considered in the therapy development.

10 Conclusion Loss of ion homeostasis plays a central role in pathogenesis of ischemic cell damage. Ischemia-induced perturbation of ion homeostasis leads to intracellular accumulation of Naþ and Ca2+ and subsequent activation of proteases, phospholipases, and formation of oxygen and nitrogen free radicals. This cascade of signal transduction events results in long-term functional and structural changes in membrane and cytoskeletal integrity and eventual cell death. Secondary active ion transport proteins including NKCC1 are important in maintaining steady-state intracellular ionic concentrations. Considerable research effort has been centered on roles of passive fluxes via cation and anion conductances in dissipation of the ion concentration gradients. Results from recent in vitro and in vivo experimental studies demonstrate the role of NKCC1 in ischemia-induced dissipation of ionic homeostasis. Therefore, the ion transport protein could be a target for the future development of therapeutic treatment of ischemic cerebral damage. Acknowledgments This work was supported in part by an NIH grant (R01NS38118). The author thanks Douglas B. Kintner for his assistance in preparation of the manuscript

Abbreviations APP CNS EAA GABA

Amyloid precursor protein Central nervous system Excitatory amino acid g-Aminobutyric acid

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[Cl–]i [Glu]i [Glu]o [Kþ]o hNKCC1 mNKCC1 MCAO NCX NCXrev NCC NKCC NKCC1 NKCC2 NKCC1þ/+ mice NKCC1þ/– mice NKCC1–/– mice OSR1 OGD PASK REOX sNKCC1 SPAK TM VSOACs WNK family of serine-threonine kinases

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Intracellular Cl– concentration Intracellular glutamate concentration Extracellular glutamate concentration Extracellular Kþ concentration Human NKCC1; KCC2, K-Cl cotransporter isoform 2 Mouse Na-K-Cl cotransporter isoform 1 Middle cerebral artery occlusion Naþ/Ca2+ exchanger Reverse mode operation of NCX Naþ-Cl– cotransporter Naþ-Kþ-Cl– cotransporter NKCC isoform 1 NKCC isoform 2 NKCC1 wild-type mice NKCC1 heterozygous mutant mice NKCC1 null mice Oxidative stress response kinase Oxygen and glucose deprivation Proline-alanine-rich STE20-related kinase Reoxygenation Shark rectal gland Na-K-Cl cotransporter STE20/SPS-1 related, Proline-alanine-rich kinase transmembrane domains Volume-sensitive organic anion channels kinases with no K = lysine, which is substituted with cysteine.

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The Naþ/Hþ Exchanger: A Target for Therapeutic Intervention in Cerebral Ischemia Jin Xue and Gabriel G. Haddad

1 Introduction The mammalian Naþ/Hþ exchanger (NHEs) family is a group of integral membrane transport proteins. It mediates an electroneutral 1:1 exchange of intracellular Hþ for extracellular Naþ and in doing so regulates intracellular pH (pHi) homeostasis and cell volume. NHEs contain two functional domains: the N-terminal transmembrane domain, which is necessary and sufficient to catalyze ion translocation, and the C-terminal cytoplasmic domain, which is crucial for modulating NHE1 activity. To date, nine isoforms have been cloned which have about 25–70% amino acid identity and are predicted to share a characteristic secondary structure. These isoforms differ in their tissue expression, subcellular distribution, kinetic properties, inhibitor sensitivity, and physiological functions. NHE1–5 are localized to the plasma membrane, though NHE3 and NHE5 can enter a recycling endosomal compartment. NHE1 is ubiquitously expressed and is considered to be a ‘‘housekeeping’’ isoform. NHE2–3 are highly expressed in the apical epithelia of kidney and intestine. NHE4 is mainly present in the stomach and basolateral epithelia of kidney. NHE5 predominantly resides in the brain. NHE6–9 are targeted to the membranes of intracellular organelles. Both NHE6 and NHE9 are present chiefly in the recycling endosomes, while NHE7 is largely in the trans-Golgi network and NHE8 is in the mid- to transGolgi pools [36, 39, 27, 47]. NHE1 is by far the most extensively studied isoform and is associated with many pathophysiological conditions. Numerous studies have shown that the activation of NHE1 during cardiac ischemia/reperfusion is deleterious. At present, there is literature on the role of NHE1 in brain ischemia and hypoxia. In this review, we focus on NHE1 and summarize recent progress in our G.G. Haddad (*) Department of Pediatrics and Neuroscience, University of California San Diego, La Jolla, CA 92093-0735; Rady Children’s Hospital, San Diego, CA 92123, USA e-mail: [email protected]


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understanding of the physiological and pathological roles of NHE1 in the brain, particularly under ischemic conditions.

2 Gene Structure The human NHE1 gene was first cloned by Sardet et al. [44]. The gene is located on human chromosome 1p36.1–p35, spans 70 kb and consists of 12 exons and 11 introns [35]. The NHE1 promoter is regulated by several transcription factors, including AP-1, AP-2, C/EBP, chicken ovalbumin upstream promoter transcription factor (COUP-TF) type I and II, as well as thyroid hormone receptor (TRa1). Moreover, a conserved poly (dA:dT) region of the NHE1 promoter is important in regulating its own expression [8]. It is worth mentioning that the NHE1 promoter is responsive to reactive oxygen species (ROS) [1]. This finding is of clinical importance because NHE1 expression/activity is elevated following ischemic disease, which may be related to the effects of ROS on the NHE1 promoter.

3 Structural Features NHE1 protein contains 815 amino acids with a calculated molecular weight of 85 kDa. But NHE1 has an apparent size of 110 kDa due to its N- and O-linked glycosylation on the extracellular loop 1 (EL1). Although NHE1 forms a stable homodimer in intact cells, individual subunits do function independently. The topological analysis has revealed that NHE1 consists of two domains: an amphipathic N-terminal membrane domain of 500 amino acids and a hydrophilic C-terminal cytoplasmic domain of 315 amino acids. The highly conserved N-terminal domain has 12 transmembrane spanning regions (TM I-XII) and 3 membrane-associated loops (intracellular loops 2 and 4 and extracellular loop 5), and is responsible for cation translocation and drug recognition. The less conserved C-terminal domain includes an ‘‘Hþ sensor’’ and serves to mediate regulation by other molecules (Fig. 1) [47, 8]. The crystal structure of Naþ/Hþ antiporter from Escherichia coli (EcNhaA) was recently elucidated [14]. Using it as a template, a 3D structure of human NHE1 is predicted. The model structure features a cluster of titratable residues that are evolutionarily conserved and are involved in the cation binding and translocation. Furthermore, an alternating-access mechanism is proposed for the transport of cations across the membrane. Low pH signal (Hþ) is probably attracted by the acidic Glu262 at the entry to the cytoplasmic funnel, then protonates Asp267. The protonation elicits a conformational change and leads to the transfer Hþ from cytoplasmic funnel to extracellular funnel. Driven by chemical gradients of Naþ and Hþ across the membrane, Hþ is exchanged for Naþ perhaps via Ser351 in the extracellular space. Finally, the movement to the alternative conformation allows the replacement of Naþ by Hþ at the cytoplasmic side. The continuance of the cycle is controlled by pHi [25].

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Fig. 1 Model of NHE1 structure. Upper panel: topology of the membrane domain which functions to transport cations. This illustration is based on the findings of [50]. Pink circles, residues implicated in both ion transport and inhibitor binding; orange circles, residues implicated in ion binding and transport; yellow circles, residues implicated in inhibitor binding; green circles, residues implicated in NHE1 folding and targeting to the plasma membrane. Lower panel: representation of the cytoplasmic domain which functions to regulate the membrane domain through interactions with signaling molecules [47] (with Journal permission)

4 Molecular Biology As illustrated in Fig. 1, the transmembrane N-terminal domain mediates cation transport and inhibitor recognition, while the cytoplasmic C-terminal domain regulates NHE1 activity and is associated with a number of functionally distinct signaling molecules. Table 1 summarizes (1) the N-terminal: the amino acids that are critical for exchanger activity, Naþ affinity, inhibitor binding/sensitivity, pHi sensing, and membrane targeting of NHE1 and (2) the C-terminal: regulatory proteins and their binding sites [36, 47, 33, 54].

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Table 1 Molecular biology of NHE1 Transmembrane (TM) N-terminal domain Function Exchanger activity Naþ affinity/inhibitor binding Inhibitor sensitivity pHi sensing Membrane targeting Cytoplasmic C-terminal domain Regulatory proteins Phosphatidylinositol 4,5-bisphosphate (PIP2) Calcineurin B homologous protein (CHP) Ezrin, radixin, moesin (ERM) family Nck-interacting kinase (NIK) Ca2þ/calmodulin Adaptor protein 14-3-3b Tescalcin Carbonic anhydrase II (CAII) Heat shock protein (HSP)70 6-phosphofructokinase-1 (PFK-1) Src homology 2 domain-containing protein tyrosine phosphatase (SHP-2)

Critical amino acids Asp159, Pro167, Pro168, Phe161 in TM IV Glu262, Asp267 in TM VII Phe161, Phe162, Leu163, Gly174 in TM IV Leu163 and Gly174 in TM IV His349 in TM IX Gly455 and Gly456 in TM XI Arg440 in IL5 Tyr454 and Arg458 in TM XI Binding sites Amino acids 513–520 and 556–564 Amino acids 515–530 Amino acids 553–564 Amino acids 538–638 Amino acids 636–656 for high-affinity site, Amino acids 657–684 for low-affinity site Amino acids 700–705 Amino acids 633–815 Amino acids 790–802 Un-determined Un-determined Un-determined

5 Tissue and Cellular Distribution NHE1 is ubiquitously expressed on the plasma membrane of virtually all mammalian cell types. In several cell types, NHE1 is not uniformly distributed along the plasma membrane but preferentially accumulates at discrete microdomains, for example, at the basolateral membrane of polarized epithelial cells, at intercalated disks and transverse tubules in cardiomyocytes, and at membrane protrusions or lamellipodia in fibroblasts [39]. We and others have demonstrated that both mRNA and protein of NHE1 are present at high levels throughout the CNS [37, 30, 7].

6 Biophysical and Electrophysiological Properties Electroneutral ion flux via NHE1 is driven by an inwardly directed gradient for Naþ and an outward gradient for Hþ, requiring no direct energy input. NHE1 exhibits a simple Michaelis–Menten dependence on extracellular Naþ, with a Km of 5–50 mM. Extracellular Liþ or Hþ competes with Naþ for the


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Naþ-binding site and extracellular Hþ suppresses NHE1 activity. In contrast, intracellular acidification allosterically stimulates NHE1 activity with a Hill coefficient of 3, which is attributed to the presence of an ‘‘Hþ-modifier site’’. During ischemia, both extracellular and intracellular pH drop, the net effect on NHE1 activity relies on the extent of extracellular inhibition versus intracellular activation. Growth factors and hormones as well as osmotic stress can activate NHE1 at normal pHi by causing alkaline shift in the set point pHi (i.e., a reduced Km for intracellular Hþ), which is believed to be at least partially dependent on protein phosphorylation/dephosphorylation [36, 39].

7 Regulatory Mechanisms: Receptorial, Transcriptional, and Transductional NHE1 is activated by a wide variety of stimuli including intracellular acidification, growth factors, hormones, cytokines, osmotic, and mechanical stresses. This activation takes place through distinct cell surface receptors including receptor tyrosine kinase, G-protein-coupled receptors, integrin receptors, and intracellular signaling networks. Modulation of NHE1 activity occurs at the COOH terminus by at least three mechanisms: (1) phosphorylation, a number of serine and threonine residues in the distal C-tail of NHE1 (amino acids 700–815) can be phosphorylated by protein kinases in response to sustained acidosis, hormones or growth factors stimulation. Such kinases include extracellular-signal-regulated kinase 1/2 (ERK 1/2), p90 ribosomal S6 kinase (p90rsk), Rho-activated kinase p160ROCK, Nck-interacting kinase (NIK), Ca2þ/calmodulin-dependent kinase II (CaMKII), and p38 mitogen-activated protein kinase (p38 MAPK). The phosphorylation results in increased NHE1 activity except for p38 MAPK, which appears to inhibit exchanger activity in response to angiotensin II. Protein kinases C and D also affect NHE1 activity but are thought not due to direct phosphorylation; (2) interaction with regulatory proteins, e.g., association with phosphatidylinositol 4,5-bisphosphate (PIP2), calcineurin B homologous protein (CHP), Ca2þ/calmodulin (CaM), adaptor protein 14-3-3b or carbonic anhydrase II (CAII) enhances NHE1 activity while with tescalcin inhibits NHE1 activity. The interaction with ezrin/radixin/moesin (ERM) is important in restrictive localization of NHE1 and plays role in structure integrity, focal adhesion, and cell migration. Src homology 2 domain-containing protein tyrosine phosphatase (SHP2)-NHE1 interaction influences NHE1 functions in pHi regulation, cell proliferation, and cell death under hypoxia; and (3) allosteric modulation via altering affinity for Hþ at ‘‘Hþ-modifier site’’ (see above Section 6) [47, 8, 33, 54]. In addition, NHE1 can be regulated at the transcriptional level (see above Section 2). Vice versa, NHE1 deficiency alters gene expression in mouse brain [57] and NHE1 activity regulates the expression of genes associated with growth factor signaling, oncogenesis, and cell cycle progression in fibroblasts [43]. In the CNS,

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ischemia/reperfusion [28, 56, 45] and epilepsy [15] elevate NHE1 expression/ activity. In contrast, chronic intermittent hypoxia downregulates the expression of NHE1 [6].

8 Physiological Properties The physiological roles of NHE1 include: (1) pHi regulation, NHE1 is activated by intracellular acidification and mediates Hþ efflux which efficiently restores pHi; (2) cellular volume regulation, NHE1 is stimulated by cell shrinkage and mediates Naþ influx, along with Cl- influx via anion exchange, which normalizes cell volume; (3) cell proliferation, NHE1-induced increase in pHi promotes G2/M entry and transition, thus facilitates cell cycle progression; (4) cell growth and differentiation; (5) an anchor for actin filament and a scaffold for the assembly of signaling complexes, roles in cytoskeleton remodeling, cell shape, focal adhesion, cell migration as well as in signal relay; (6) apoptosis, role of NHE1 differs depending on stimulation and cell type; and (7) a possible mediator of immunity [36, 39, 33, 5].

9 Pathophysiological Relevance in Stroke Cerebral ischemia is a complex pathological process, which results in energy failure, cell swelling, membrane depolarization, abnormal release of excitatory neurotransmitters and Kþ ions, spreading depression, injurious elevation in [Naþ]i and [Ca2þ]i, generation of free radicals, blood–brain barrier (BBB) disruption and edema, as well as an inflammatory response. Ionic disturbance, particularly for Hþ, Naþ, Caþ2, and Kþ, is an early event in the cascade leading to hypoxic injury in the mammalian brain. NHE1, a major regulator of pHi and ionic homeostasis, has been implicated in ischemia/reperfusion injury. NHE1 is widely distributed in neurons, glial cells, BBB endothelial cells, and choroid plexus [39]. Because of its high metabolic rate, the CNS produces a large amount of metabolic acid, particularly under stressful conditions, such as hypoxia/ischemia. This feature renders neurons/glial cells more vulnerable to injury from acidosis. Therefore, the efficient acid-extrusion mechanism is crucial for proper brain function. The growing evidence indicates that NHE1 is a key player in pHi regulation in both neurons and glial cells under steady-state conditions and after intracellular acidification [40]. Consistent with this view, acutely dissociated CA1 neurons from NHE–/– mice or from NHEþ/+ mice treated with NHE1 inhibitor HOE694 exhibit reduced steady-state pHi and reduced recovery rate from acid load [55]. Similar results have been reported in primary cortical neurons and astrocytes either with inhibitor or from genetic ablation [29, 20]. Both beneficial and detrimental effects of acidic pH (‘‘pH paradox’’) have been described in the brain [39]. Mild acidosis is protective in


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cerebral ischemia by attenuating Ca2þ influx, by reducing energy demand and ATP depletion [48], and by inhibition of NHE1 via acidic pHo [49]. However, if pHi is too low, cell death ensues due to: (1) depolarization, over-excitability and oxygen consumption; (2) potentiation of free radical formation; (3) inhibition of mitochondrial metabolism and glycolysis, thus accelerating ATP depletion; and (4) enhancement of the neurotoxicity mediated by -amino-3-hydroxy-5methyl-4-isoxazole propionic acid receptors [52]. Ischemia/hypoxia stimulates NHE1 by a reduction in pHi or via signaling pathways (e.g., ERK-p90RSK or PKA or PKC) [28, 56, 45, 18]. In acutely isolated mouse CA1 neurons, 5 min of chemical anoxia elicits pHi elevation in HEPES buffer, which can be reduced in NHE1 null mice, or by NHE1 antagonist 3-methylsulfonyl-4-piperidinobenzoyl-guanidine methansulfonate (HOE694), or inhibitors of PKA or PKC, indicating that anoxia activates NHE1 acutely, probably via PKA or PKC (Fig. 2) [56]. Similarly, in rat hippocampal CA1 neurons, NHE1 is stimulated immediately after anoxia via cAMP–PKA signaling, yet is apparently inhibited during the anoxia phase,

Fig. 2 NHE1 contributes to anoxia-induced pHi changes in mouse CA1 neurons. (1) Effect of HOE-694 on the anoxia-induced pHi change in HEPES buffer. A: With 100 mM HOE-694 in HEPES buffer, 5 min of anoxia induced no change in pHi in contrast to an increase in pHi in controls. B: Mean changes of pHi in control and the HOE-694 group. Means are significantly different from each other. (2). Anoxia-induced pHi increase was smaller in the NHE1 null mutant neurons. A: Two neurons (one was a wild-type (WT) and the other a NHE1 mutant) with similar initial pHi. Both neurons were perfused with HEPES buffer and subjected to 5 min of anoxia. Although both neurons had a similar pattern in pHi change in response to anoxia, a smaller increase was seen in the mutant neuron. B: Mean change of pHi induced by 5 min of anoxia in both NHE1 mutant and wild-type neurons. Means are significantly different from each other [56] (with Journal permission)

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Fig. 2 (continued)

possibly due to a fall in cellular ATP level [46]. However, OGD/REOX (oxygen and glucose deprivation/re-oxygenation)-induced increase in NHE1 activity is independent of prolonged acidosis but depends in part on the activation of ERK1/2 in cultured cortical neurons or astrocytes [28, 18]. The role of NHE1 in cerebral ischemia has been examined in two types of in vitro ischemic models, the hypoxic, acidic, ion-shifted Ringer’s solution (HAIR) or OGD/REOX. HAIR solution simulates interstitial ionic changes of cerebral ischemia. After a 20–40-min exposure to HAIR, most astrocytes die within 30 min upon return to normal saline (i.e., ‘‘reperfusion’’). Astrocyte death requires external Ca2þ and is blocked by 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl] isothiourea methanesulfonate (KB-R7943), an inhibitor of reverse Naþ–Ca2þ exchange (NCX), suggesting that injury is triggered by a rise in [Ca2þ]i. Moreover, the rise of [Ca2þ]i and injury are prevented by NHE inhibitors ethyl isopropyl amiloride and HOE-694. Acidic reperfusion media, which inhibit NHE1 activity, is also protective. These data indicate that Naþ loading via NHE1 fosters reversal of NCX and subsequent cytotoxic elevation of [Ca2þ]i, which eventually causes ischemic damage of astrocytes [19, 3]. In agreement with this idea, OGD/REOX evokes a 7-fold and 1.5fold increase in [Naþ]i and [Ca2þ]i, respectively in cortical neurons, accompanied by mitochondrial Ca2þ accumulation, cytochrome c release and neuronal death. These deleterious changes can be attenuated by NHE1 inhibitor (4-Isopropyl-3methylsulfonyl-benzoyl)-guanidine methanesulfonate (HOE642) or genetic ablation of NHE1 activity or 2-[4-[(2,5-difluorophenyl) methoxy]phenoxy]-5-ethoxyaniline (SEA0 400, a potent blocker of reversal mode of NCX), confirming again the importance of the above-mentioned injury mechanism (Fig. 3) [29].


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Fig. 3 Inhibition or ablation of NHE1 activity abolishes OGD-mediated cell death. Cell mortality was assessed in 12–15 DIV cortical neurons after 3 h of OGD and 21 h of REOX. For HOE 642 treatment, NHE1þ/+ neurons were incubated in the presence of 1 mM HOE 642 at 378C for 3 h of OGD and 21 h of REOX. Sister NHE1þ/+ cultures were incubated for 24 h in normoxic control buffers (Con). Similar assays were also performed in NHE1þ/– and NHE1–/– cultures. At the end of the experiment, cells were stained with PI and calcein-AM, and cell images were acquired. A, C, E, Calcein-AM. B, D, F, PI. A, B, Con. C–F, OGD/ REOX. E, F, OGD/REOX plus HOE. Scale bar, 256 mm. G, summary data. Data are means SD (n = 5–8 cultures). *P < 0.05 versus Con; #P < 0.05 versus NHE1þ/+ OGD/REOX. Error bars represent SD [29] (with Journal permission)

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Cerebroprotection by NHE1 blockade has been validated in an in vivo ischemic model (middle cerebral artery occlusion (MCAO)). A marked decrease of infarct volume is found in NHE1þ/– mice or HOE642-treated NHEþ/+ mice following 1-h focal ischemia (Fig. 4) [29]. Other NHE1 inhibitors, N-(aminoiminomethyl)-1-methyl-1 H-indole-2-carboxamide methanesulfonate (SM-20220), 3-(2,5-dichlorothiophen-3-yl)-N-[2-(dimethylamino)ethyl]-5-guanidinocarbonylbenzamide dihydrochloride (FR183998), and N-carbamimidoyl-4-[4-(1Hpyrrol-2-ylcarbonyl)piperazin-1-yl]-3-(trifluoromethyl) benzamide (sabipor-

Fig. 4 Inhibition or knockdown of NHE1 activity reduces infarction after focal ischemia. (1) Inhibition of NHE1 activity. A, B, After 2 h of MCAO and 24 h of reperfusion, saline-treated control and HOE 642-treated NHE1þ/+ mice were killed. Infarction volume (outlined area) was determined by TTC staining. Data are means SD (n = 4–6). *P < 0.05 versus NHE1þ/+. Scale bar, 5 mm. C, focal ischemia induced a significant increase in water content. *P < 0.01 versus contralateral. Error bars represent SD. (2) Knockdown of NHE1 activity. A, crude cortex membrane proteins of three genotypes were separated electrophoretically. Expression of NHE1 and tubulin III in NHE1þ/+, NHE1þ/–, and NHE1–/– mouse cortex was shown on the same blot. Densitometric analysis of immunoblots was presented as a ratio of NHE1/-tubulin III band intensity. Data are means SD (n = 3–5). B, C, After 2 h MCAO and 24 h reperfusion, infarction volume in NHE1þ/+ or NHE1þ/– mice was determined by TTC staining. *P < 0.05 versus NHE1þ/+. Data are means SD (n = 4). Scale bar, 5 mm. D, water content in NHE1þ/ + and NHE1þ/– mice after 2 h of MCAO and 24 h of reperfusion. Data are means SD (n = 4–6). *P < 0.01 versus contralateral. Error bars represent SD [29] (with Journal permission)


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ide), have also been proven to provide similar beneficial effects in MCAO model [22, 11, 21, 38]. Most importantly, SM-20220 [24] and sabiporide [38] show effectiveness even with post-ischemia administration, emphasizing their therapeutic potential for stroke. Brain edema is another characteristic of cerebral ischemia. It is formed during early hours of ischemia and by a process involving increased secretion of Naþ and water across an intact BBB with a concomitant increased uptake of Naþ, Cl–, and water into astrocytes which cause cytotoxic edema. NHE1 along with Naþ-Kþ-2Cl– cotransporter isoform 1 (NKCC1) contributes to both BBB secretion and astrocyte-uptake processes [17]. Supporting this notion is that NHE1 inhibition significantly alleviates cellular swelling induced either by OGD/REOX [29, 20] or glutamate [32] in cultured cortical neurons or astrocytes. Moreover, reduction on the extent of cerebral edema by NHE1 blockade is observed in an in vivo ischemic model (MCAO) [22, 21, 38]. Cerebroprotective benefits of NHE1 inhibition is manifested further by the facts that NHE1 inhibition: (1) attenuates leukocyte adhesion [10] and improves endothelial dysfunction [12] induced by ischemia-reperfusion; (2) facilitates consciousness recovery and preserves neurological function after transient cerebral ischemia [23]; (3) reduces the magnitude of the ischemia-induced increase in locomotor neuronal activity, which represents the degree of damage of hippocampal CA1 neurons [41]; (4) protects against excitotoxicity [38, 32]; and (5) prevents ischemia/reperfusion-elicited free fatty acid release [42]. Spontaneous [4] and targeted [2] NHE1–/– mice suffer from epileptic-like seizure, ataxia, growth retardation, and selective neuronal death in the cerebellum and brainstem, suggesting that NHE1 is important in neuronal function, growth, and survival. Mice lacking NHE1 have been shown to (1) upregulate Naþ channel expression in hippocampal and cortical regions, leading to an increase in Naþ current density and neuronal excitability [9, 51]; (2) change the expression of other membrane transporters at both protein and mRNA levels [53]; and (3) alter the expression of genes implicated in neurodegeneration [57]. These findings may help us understand the molecular basis of the phenotypical changes in NHE1 null mutant and potentially why they seize. Systemic administration of NHE1 inhibitors is associated with a higher rate of cerebrovascular events [34] and shows neurotoxic effects [13] with the latter being reversible by 7 days after treatment. Clearly, future investigation is needed to delineate the precise mechanism of the pertinent adverse effects in order to dissociate such unwanted side effects from the cerebroprotective efficacy of NHE1 inhibitors.

10 Pharmacological Modulation Pharmacological inhibitors of NHE1 are categorized into three groups: (1) amiloride and its analogues; (2) benzoylguanidine and its derivatives; and (3) bicyclic guanidines. Amiloride, a Kþ-sparing diuretic, was the first drug

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described as a NHE1 inhibitor, which also inhibits a conductive Naþ channel, Naþ/Ca2þ exchanger, and Naþ/Kþ ATPase. Double substitution of the nitrogen of the 5-amino group of amiloride gives dimethylamiloride (DMA), 5-(Nethyl-N-isopropyl)-amiloride (EIPA), 5-(N-methyl-N-isobutyl)-amiloride (MIBA), and 5-(N, N-hexamethylene) amiloride (HMA). They are much more effective than amiloride but weak selective toward NHE1. The simultaneous replacement of the pyrazine ring by a phenyl and of the 6-chloro by a sulfomethyl leads to drugs such as HOE-694, HOE642, 2-methyl-5-methylsulfonyl-4-(1-pyrrolyl)-benzoylguanidine methanesulfate (eniporide), and benzamide-N-(aminoiminomethyl)-4-[4-(2-furanylcarbonyl)-1-piperazinyl]-3(methylsulfonyl)methanesulfonate (BIIB-513), which have high potency/selectivity for NHE1, excellent solubility, resorption, and bioavailability. In the last decade several molecules based on the bicyclic template have been designed, including [1-(quinolin-5-yl)-5-cyclopropyl-1H-pyrazole-4-carbonyl] guanidine (zoniporide), SM-20220, N-(aminoiminomethyl)-1,4-dimethyl-1H-indole-2carboxamide methanesulfonic acid (SM-20550), (1R,3R)-N-(Diaminomethylene)-3-(2,3-dihydro-1-benzofuran-4-yl)-2,2-dimethylcyclopropanecarboxamide (BMS-284640), (5E,7S)-[[7-(5-fluoro-2-methylphenyl)-4-methyl-7,8-dihydro-5(6H)-quinolinylidene]amino]guanidine dimethanesulfonate (T-162559), 6,7,8, 9-tetrahydro-2-methyl-5H-cyclohepta[b]pyridine-3-carbonylguanidine maleate (TY-12533), or 3-[(cyclopropylcarbonyl)amino]-N-[2-(dimethylamino)ethyl]-4-[4-(5-methyl-1H-imidazol-4-yl)piperidin-1-yl]benzamide (SL 59.1227). The IC50 for the human NHE1 are as follows: Amiloride = 10.7 mM, Cariporide = 0.08 mM, T-165229 = 13 nM. Both chemical structure and ionization of the guanidine determine the potency of NHE inhibitors. During ischemia/ reperfusion, the pH can fall down to 6.2 or lower, drugs like cariporide (pKa = 6.28), TY-12533 (pKa = 6.93) or zoniporide (pKa = 7.2), which are positively charged, become more efficient. Indeed, cariporide and TY-12533 are more active at pH 6.2 than 6.7 (cariporide: IC50 = 22 nM/120 nM; TY-12533: IC50=17 nM/32 nM). In an aqueous medium, Naþ ion is surrounded by three molecules of H2O and guanidinium ion of inhibitors is similar in structure, thus can block NHE1 by competitive binding to the extracellular Naþ-binding site [26, 31].

11 Preliminary Clinical Trials Extensive preclinical studies have demonstrated that NHE1 inhibition affords excellent and consistent protection from myocardial ischemia/reperfusion injury in a wide variety of experimental models and animal species by different investigators [16]. However, in human, clinical trials have resulted in only modest success [34], i.e., a significant reduction in the combined endpoint of death and myocardial infarction in high-risk patients undergoing coronary artery bypass graft surgery. Unfortunately, this beneficial effect is associated with a higher


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rate of cerebrovascular events. The underlying mechanisms for increased cerebrovascular risk are unknown. Further investigation is needed to determine whether it reflects NHE1 inhibition in general or whether this is a specific effect of a particular inhibitor. Our current knowledge regarding the role of NHE in cerebral ischemia is limited, though emerging evidence points to cerebroprotective effects of NHE inhibition. More evaluation is warranted in animal models of cerebral ischemia before devising clinical trial.

12 Therapeutic Perspectives Stroke is the second-most prevalent cause of death worldwide. About 80% of all strokes are ischemic. Despite years of research efforts, the effective treatment and prevention of ischemic brain injury remains a major medical challenge. NHE1 inhibition with either pharmacological agents or genetic ablation has been reported to reduce brain damage after ischemia/reperfusion insult, both in vitro culture and in vivo animal models. These encouraging findings hold promise for a new drug that could significantly reduce injury associated with cerebral ischemia.

13 Conclusions In this review, we have documented the research progress in understanding the physiopathological roles of NHE1 in the CNS. Although NHE1 is pivotal for maintaining pHi and ionic homeostasis under physiological stresses, its excessive activity, such as following cerebral ischemia, may be a major cause of cell damage and death. The possible injury mechanisms by ischemia-induced NHE1 activation include (1) [Naþ]i accumulation leading to NCX reversal, elevated [Ca2þ]i, and consequent cell death that may involve mitochondrial death pathway; (2) changes in pHi; (3) brain edema and cell swelling; (4) activation of MAPKs; and (5) release of excitatory amino acids or ROS [39]. Cerebroprotective roles of NHE1 inhibition have been demonstrated in in vitro and in vivo ischemic models, making it an attractive therapeutic target for cerebral ischemia. However, adverse effects by NHE1 inhibitors have also been noted. Thus, better understanding of the underlying mechanisms both for protection and for side effects should be future research focus.

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The Naþ/Kþ-ATPase as a Drug Target for Ischemic Stroke Melissa A. Gottron and Donald C. Lo

The Naþ/Kþ-ATPase (also known as the ‘‘sodium–potassium pump’’ or just ‘‘sodium pump’’) is a ubiquitous and critically important protein complex in the human body. In the brain, between 40 and 70% of all energy is spent maintaining its ionic transport activity [1, 2, 3]. This energy is used for the active exchange of cytosolic sodium for extracellular potassium in a 3:2 ratio, a process required for the maintenance of transmembrane ionic gradients for all mammalian cells, which in turn is essential for setting the cellular resting potential, regulating osmolarity, and powering the secondary transport of other important solutes like calcium (via the sodium–calcium exchanger) and protons (via the sodium–hydrogen exchanger). While these functions are especially critical for neuronal signaling (notably action potentials and synaptic transmission) and blood filtration in the kidney, all cells in the body ultimately depend on the Naþ/Kþ-ATPase to support their fundamental membrane processes. As the brain’s primary consumer of ATP, the sodium–potassium pump is particularly vulnerable to ATP depletion common in ischemic stroke. This vulnerability suggests that pharmacological inhibition of the Naþ/Kþ-ATPase would further compromise ATP-depleted neurons, yet there is accumulating evidence that inhibiting the sodium–potassium pump can actually provide neuroprotection in the context of ischemia as well as other traumatic and neurodegenerative conditions. The present chapter will thus review several aspects of the basic properties of the Naþ/Kþ-ATPase, mechanisms responsible for regulating Naþ/Kþ-ATPase function, and recent studies implicating the Naþ/Kþ-ATPase in ischemic stroke.

D.C. Lo (*) Center for Drug Discovery and Department of Neurobiology, Duke University Medical Center, Durham, NC, USA e-mail: [email protected]


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1 Basic Properties 1.1 Gene Structure The Naþ/Kþ-ATPase was the first ionic transporter to be discovered and remains the focus of much important research more than 50 years later [4]. Like the hydrogen–potassium and calcium pumps, it is a member of the P-type ATPase protein family [5]. Members of this family earn their name from the gamma phosphate group that controls their activation; they become autophosphorylated by the ATP they hydrolyze, and this phosphorylation catalyzes conformational changes essential for pump function. Although all P-type ATPases share a common structure containing a heterodimeric protein core, significant functional differences arise from variations in the specific composition of this general structure. The Naþ/Kþ-ATPase consists of a heterodimeric core of a and b subunits that may be accompanied by a third g subunit [reviewed in 6, 7, 8]. These subunits are assembled in the endoplasmic reticulum before transport to the plasma membrane, and vectorial Golgi sorting as well as stabilization mechanisms play important roles in the pump’s targeted delivery [9, 10]. Both the a and b subunits are obligatory for the pump’s proper assembly and function [reviewed in 11]. The different subunits of the Naþ/Kþ-ATPase have distinct properties with respect to its overall functions (see Fig. 1). The a subunit of the Naþ/KþATPase is a 112 kDa protein with 10 membrane-spanning units whose N and C termini are intracellular. Because this subunit contains sites important for

Fig. 1 General structure of the sodium–potassium pump. The pump is a transmembrane protein composed of two major subunits that transport sodium and potassium between the cytoplasm and the extracellular milieu in a 3:2 ratio. While the a subunit is crucial for ATP binding and phosphorylation, ion occlusion, and catalytic activity, the b subunit plays a key role in pump maturation, stabilization, and transport. Both subunits are influenced by pharmacological modulation of the pump.

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ATP binding and phosphorylation as well as ion occlusion, it forms the catalytic core of the pump. The a subunit is also the primary binding site for many pharmacological agents that affect pump activity. Although their unique functional roles are still poorly understood, four isoforms of the a subunit are known to exist. The a1, a2, a3, and a4 isoforms differ in their kinetics, affinity for different ligands, and tissue and cellular distribution. Non-redundant functions for the different subunit isoforms are suggested by the findings that (1) although the a1 isoform is alone sufficient for the resting ion-transfer state, it cannot compensate for a lack of a2 during conditions of greater pump activity [12], (2) mutants lacking a2 and a3 isoforms show deficits in learning and motor activity as well as symptoms of anxiety [13], and (3) mutations of the gene encoding the a2 isoform have been associated with migraine disorders in humans [14]. The b subunit is a 35-kDa protein with a single, type II transmembranous region. This glycosylated subunit plays a key role in the maturation, transport, and stabilization of the pump as it migrates from the endoplasmic reticulum to the plasma membrane, and it also contains key residues necessary for potassium occlusion and ouabain binding [15, 16]. As for the a subunit, multiple b subunit isoforms (b1, b2, and b3) differentially influence Naþ/Kþ-ATPase affinity and stability. The g subunit, a member of the FXYD family, is a 6.5-kDa type I membrane protein associated with the Naþ/Kþ-ATPase in a tissue-specific manner. While not essential for basic pump expression or activity, the g subunit is typically present in a 1:1 molar ratio with the a and b subunits and interacts with the C-terminus of the a subunit in certain tissues [17, 18, 19]. This subunit modulates both the function and distribution of the pump by increasing its affinity for ATP [20, 21, 22]. Other members of the FXYD protein family that show a high degree of homology to the g subunit, like phospholemman [23], channelinducing factor [24], and mammary tumor-associated 8-kDa protein [25], have similarly been suggested to interact with the Naþ/Kþ-ATPase in various nonneural tissues [26].

1.2 Tissue/Cellular Distribution The importance of the Naþ/Kþ-ATPase to cellular function is highlighted by the fact that it is present in every cell of the body. Although a single cell may express more than one a or b isoform [27], the expression of different a and b isoforms is regulated in both a developmental and tissue-specific manner [28, 29, 30]. Tissue Distribution. The a1, a2, and a3 (but not a4) isoforms of the Naþ/KþATPase are expressed in the brain [31, 32]. While the a1 isoform is ubiquitously expressed in the central nervous system, the a2 and a3 isoforms show more restricted neural expression patterns [27, 28, 30, 33, 34]. The a2 isoform is widely

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expressed in neurons during development [28, 34] but becomes confined primarily to glia and a few types of neuron in the adult brain [27, 30]; in contrast, the a3 isoform shows primarily neuronal expression in the adult [27, 30]. Similar to the a subunit, the three b subunit isoforms are expressed to varying degrees in the brain. Pumps with the most common isoform, b1, are expressed ubiquitously throughout the body [33, 35]; those with b2 are largely found in excitable tissues like the heart and brain [36]; and those with b3 show only minimal neural expression [37]. The b isoforms show cell-specific mRNA [30] and protein expression [33, 38, 39], with the b1 and b2 isoforms expressed primarily in neurons and glia, respectively. Finally, the isoform composition of sodium–potassium pumps in different parts of the brain appears to be both complex and idiosyncratic [33, 38]. Neither preferential association of different a and b isoforms nor neurotransmitter-specific pairings has been observed. Due to their differing affinities for ligands, however, different isoform pairings confer unique functional properties to the cell types in which they are expressed. Subcellular Distribution. The a and b isoforms of the sodium–potassium pump can be targeted to specific subcellular locations within neurons and glia [39, 40, 41]. Although the distribution of a1 isoform is relatively uniform throughout the plasma membrane [42, 43], knockout studies have suggested that interactions between the a1 isoform and the IP3 receptor are functionally important [44]. In contrast, interactions with the cytoskeletal proteins ankyrin, spectrin, actin, and adducin preferentially target the a2 and a3 isoforms to specialized lipid rafts in the plasma membrane overlying the endoplasmic reticulum [42, 43, 45]. Because these locations are also enriched with the sodium–calcium exchanger, this places the a2 and a3 isoforms in an ideal situation to regulate the local concentrations of sodium and calcium in the cytosolic space between the plasma membrane and endoplasmic reticulum [46]. The subcellular localization of the pump thus permits specific functional control over cellular processes.

1.3 Biophysical Properties A well-accepted general description of the pump’s electrogenic activity is given by the Albers-Post scheme [47, 48, 49]. In the Albers-Post model, the pump alternates between two states (E1 and E2) that differ in both conformation and ion-binding affinity. Transitions between the E1 and E2 states are dependent on both voltage as well as local concentrations of sodium, potassium, and ATP, and these transitions are accompanied by the successive transport of sodium ions out of and potassium ions into the cell. In the Albers-Post model, the resting pump exists in the E1 conformation (Fig. 2). This conformation opens the Naþ/Kþ-ATPase’s cationbinding pocket to the intracellular space, which allows it to bind three


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intracellular sodium ions and one molecule of ATP. Hydrolysis of the bound ATP permits phosphorylation of pump and leads to the occlusion of the three sodium ions. A spontaneous conformational change then causes the pump to assume the E2 conformation, which opens toward the extracellular space. Because this conformation has a low affinity for sodium, the Naþ/Kþ-ATPase releases the occluded sodium ions into the extracellular space to complete the outward transport of sodium ions. The complementary inward transport of potassium begins with the exchange of sodium with potassium in the pump’s phosphorylated E2 conformation (Fig. 2). The extracellular binding of two potassium ions is followed by the spontaneous dephosphorylation of the E2 conformation, which results in the occlusion of the two potassium ions. Upon relaxation of the pump to the E1

Fig. 2 Albers-Post model of sequential ion transport. The Albers-Post model describes a potential mechanism for the exchange of sodium and potassium. The resting pump exists in the E1 state facing the cytoplasm. ATP as well as three sodium ions bind to the pump, and hydrolysis of the ATP permits phosphorylation of the pump and occlusion of the bound sodium ions. A spontaneous change in conformation allows the pump to assume the E2 conformation, which opens toward the extracellular space and releases the bound sodium into the extracellular space. The transport of potassium begins with the binding of two potassium ions to the pump’s phosphorylated E2 state. The spontaneous dephosphorylation of the E2 conformation occludes the two bound ions. When the pump relaxes to the E1 conformation, the potassium ions dissociate from their binding pocket and are released into the cytoplasm.

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conformation, the binding pocket containing the potassium ions opens into the cell. The binding of ATP inside the cell stimulates the release of the potassium ions and thus begins a new catalytic activity cycle. The Albers-Post scheme implies that a single a/b heterodimer is sufficient for activity of the Naþ/Kþ-ATPase, because ions are successively transported within a shared binding pocket. However, it should be noted that other schemes in which the Naþ/Kþ-ATPase functions as a higher-order oligomer have also been proposed [50, 51, 52]. Due to its role in the transport of sodium and potassium in a 3:2 ratio, the Naþ/Kþ-ATPase is electrogenic and works to hyperpolarize the cell. This active process requires both the consumption of O2 and hydrolysis of ATP [7, 53]. When either of these substrates is not available (as in ischemic stroke), pump failure produces a defined set of intracellular changes that includes the decrease of intracellular potassium, increase in intracellular sodium, and depolarization of the membrane [54]. These changes affect numerous intracellular processes, promulgating the damage caused by ATP scarcity.

1.4 Structural Features Although a great deal of structural similarity exists among the P-type ATPases, the Naþ/Kþ-ATPase is unique in that it is the only cation transporter with highaffinity binding sites for both potassium and ATP. While the molecular composition of the ion-binding pockets of the pump has spurred debate, numerous molecular biology studies were recently confirmed by the solution of a crystal structure describing the ion binding pockets of the Naþ/Kþ-ATPase [55]. The crystal structure identified a potassium-binding region between the fourth, fifth, and sixth transmembrane regions of the a subunit that simultaneously holds both potassium ions. Interestingly, two presumed sodium-binding sites overlap significantly with this potassium-binding pocket. Because such a shared binding pocket suggests sequential ion transport, this finding supports the Albers-Post sequential transport model described above. The location of the third sodiumbinding site remains unclear, but a distinct binding site may exist in the C-terminus of the a subunit. Other interactions of the Naþ/Kþ-ATPase’s functional domains have also been identified. The cytoplasmic loop between transmembrane segments four and five of the a subunit is essential for ATP hydrolysis, and regions near the seventh and ninth transmembrane segments are critical for interactions with the b and g subunits, respectively. These latter interactions differ sterically in that the b subunit is thought to completely cover the cytoplasmic loops between transmembrane segments 5–6 and 7–8 in the a subunit like a lid, whereas the g subunit is thought to intercalate between the a and b subunits. These findings are remarkably consistent with those implied by earlier molecular biology and modeling studies [16, 56, 57, 58].


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Finally, the pharmacological agents discussed in detail later in the chapter affect the pump through actions at specific locations. Cardiac steroids prevent pump activation by binding within a groove between the extracellular loops of the a subunit that permits its interaction with the b subunit, and mutagenesis studies have identified a number of amino acid residues critical for this interaction [59, 60, 61]. It is interesting and clinically relevant that the cellular ionic environment can influence the efficacy of pump blockade: high potassium levels lower the affinity of the Naþ/Kþ-ATPase for pharmacological blockers whereas sodium inhibits this effect [7]. Finally, although the binding sites for other drugs affecting pump activity are known to be distinct from those of the cardiac steroids, their precise locations remain elusive [62].

2 Regulation 2.1 Specific Pharmacological Inhibition Because of its pivotal role in cellular function, it is not surprising that the Naþ/ Kþ-ATPase is the target of a host of natural toxins derived from both plants and animals. These toxins typically stop the pump from functioning either by blocking its ion transport activities or by holding it ‘‘open’’ such that it resembles an open ionic channel. Both of these changes critically impair the maintenance of proper sodium and potassium gradients within the cell. Toxins that block the pump’s ion transport activities include ouabain and the cardenolide cardiac glycosides. As noted above, these toxins reversibly bind to the extracellular face of the Naþ/Kþ-ATPase in the phosphorylated E2 conformation. Cardiac glycosides consist of two main structural moieties: the aglycone moiety contains an R-group that defines the cardiac glycoside’s class and is important for its function, whereas the sugar moiety does not greatly influence biological activity but likely plays a role in the targeting and modulation of the drug. These drugs are toxic at high doses due to their blockade of pump activity, which leads to rundown of the sodium and potassium gradients, membrane depolarization, and secondary dysregulation of other ionic gradients. However, in lower doses the cardiac glycosides have been clinically effective and have been in therapeutic use since the eighteenth century in treating congestive heart failure [63](see Section 3.1). Palytoxin and sanguinarine, derived from the marine coral Palythoa toxica [64] and the plume poppies Macleaya cordata [65], respectively, are the primary members of a second class of Naþ/Kþ-ATPase toxins. Members of this class bind extracellularly to the phosphorylated E1 conformation of the Naþ/KþATPase to lock the pump in an open conformation [66]. This essentially converts the pump into a low-conductance ion channel, permitting a 10 pS current carried by potassium ions to flow out of the cell [64, 67]. The resulting rundown

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of sodium and potassium gradients across the cell membrane is extremely toxic and, indeed, palytoxin is perhaps the most toxic substance known – just 4 mg constitutes a lethal dose for humans.

2.2 Intrinsic Regulatory Mechanisms Because of its expression in every cell of the body, it is hardly surprising that multiple mechanisms exist to regulate the Naþ/Kþ-ATPase. These regulatory mechanisms are particularly important in cells, like neurons or those in nephrons, that undergo large fluctuations in ionic content due to neural activity or changes in diet and exercise. To ensure proper functioning, these cells must be able to titrate Naþ/Kþ-ATPase activity quickly and precisely in response to a variety of environmental challenges. That the Naþ/Kþ-ATPase operates at just 50% of its maximum capacity at rest implies that it can respond to a wide range of physiological stimuli by changing its activity level. The global activity of the Naþ/Kþ-ATPase within a cell can be changed either by (1) altering the number of pumps in the membrane or (2) altering the activity of individual pumps already existing within the membrane. Signaling pathways leading to both of these types of changes via modulation of Naþ/KþATPase transcription, transport, and functional kinetics have been described downstream of interactions with a variety of membrane proteins and hormones [reviewed in 68]. Regulation of pump number. Changes in the complement of Naþ/Kþ-ATPases in the membrane can arise through transcriptional, transport, and membrane recycling mechanisms. Transcriptional control of the Naþ/Kþ-ATPase was initially suggested by the observation that Naþ/Kþ-ATPase subunit transcription increases when the pump is blocked pharmacologically [69]; later work indicated that the 50 -flanking sequence of the gene encoding the a subunit contains both positive and negative transcriptional regulatory sites [70]. Cascades involving protein kinase A (PKA), extracellular signal-regulated kinase 1/2 (ERK1/2), and histone deacetylase have been implicated as critical regulators of a subunit transcription, whereas protein kinase C (PKC), c-Jun-N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) have been implicated as critical regulators of b subunit transcription [71]. Other brain-derived substances, such as dopamine and nitric oxide, also influence the transcription of the Naþ/Kþ-ATPase via activation of different signaling cascades [72, 73]. Finally, manipulations altering extracellular calcium concentrations affect the transcription and degradation rates of both the a and b subunits [69]. Changes in the transport of the Naþ/Kþ-ATPase from the ER also influence the complement of Naþ/Kþ-ATPase that accumulates in the plasma membrane. Many proteins are essential for proper targeting of the Naþ/Kþ-ATPase to specific domains within the plasma membrane, and cascades leading to the activation or inhibition of these proteins thereby influence Naþ/Kþ-ATPase activity. For example, arrestin and spinophilin compete to regulate the


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trafficking of the Naþ/Kþ-ATPase via interaction with its cytoplasmic loop [74]; activity promoting the dominance of one or another of these proteins therefore influences the complement of pumps at the membrane surface. Similarly, cascades that alter the presence or activity of the cytoskeletal proteins important for correct targeting of the pump to plasma membrane subdomains (e.g., ankyrin and spectrin) also influence overall Naþ/Kþ-ATPase levels and activity at the membrane. Finally, alterations in the rates at which the Naþ/Kþ-ATPase is either endoor exocytosed from the membrane rapidly affect the net complement of functional pumps available to transport sodium and potassium. Dopamine, RhoA, and the AMP kinases have all been shown to increase the rate of Naþ/KþATPase endocytosis from the membrane, thereby reducing its activity [68, 75, 76]. Other mechanisms of Naþ/Kþ-ATPase regulation, including that by IL-1, work by destabilizing the actin cytoskeleton and redistributing the pump via membrane recycling [77]. Regulation of pump activity. The second general class of Naþ/Kþ-ATPase regulation involves altering how effectively pumps already present in the membrane transport ions. This can be accomplished indirectly via restriction of the rate-limiting factors required for the pump’s activity or by direct interference with the pump via phosphorylation or protein binding. The most straightforward mechanisms of pump regulation involve varying the concentrations of its three major substrates: sodium, potassium, and ATP. The Naþ/Kþ-ATPase is normally most sensitive to changes in the concentration of intracellular sodium, because its half-maximal activation occurs at steady-state sodium levels [78]. This means that variations due to the activation of other sodium transporters greatly influence Naþ/Kþ-ATPase activity. In contrast, potassium is typically in excess under resting conditions and affects Naþ/Kþ-ATPase activity only to the extent that it competes with sodium for access to the pump [79]. Finally, while most cells contain adequate stores of ATP to power the Naþ/Kþ-ATPase, the binding of ATP is critical regulator of pump activity in cells with low ATP levels due to ischemia [80]. In addition to these changes in catalytic activity, Naþ/Kþ-ATPase stability is also heavily modulated by the cellular ionic environment [81]. The pump’s activity is modulated by direct interference with the pump itself as well. In fact, endogenous cardiotonic steroids have been discovered that are highly regulated under physiological and pathophysiological contexts and that inhibit the activity of the Naþ/Kþ-ATPase [reviewed in 82, 83]. The structure of these endogenous inhibitors is almost identical to that of the plant-derived Naþ/ Kþ-ATPase inhibitor ouabain. They are released from the adrenal gland and midbrain in response to a variety of environmental stimuli, and appear to interact with a specific hydrophobic binding partner that may influence their tissue distribution and inhibitory effects on ion transport [82]. Finally, modulation of Naþ/Kþ-ATPase activity can occur via phosphorylation/dephosphorylation by such proteins as PKA, PKC, PP2B, PKG, and others [reviewed in 68]. The idiosyncratic effects of phosphorylation on pump

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activity are both tissue- and species-specific, so that the activation of a particular signaling cascade may inhibit the pump in certain circumstances while increasing its activity in others. Environmental stimuli exert their effects through such signaling pathways in regulating the function of the Naþ/Kþ-ATPase. It is relevant for the current discussion of stroke that both hypoxia and hyperoxia can decrease the activity of the Naþ/Kþ-ATPase [84]. Although some such inhibition is likely downstream of ATP depletion due to improper oxygenation of mitochondria, proximal causes of decreased Naþ/Kþ-ATPase activity also include phosphorylation [85], free radical-induced damage [86], altered rates of endocytosis [85, 87], and changes in the complement of a and b subunit isoforms [88]. The physiological implications of such Naþ/Kþ-ATPase downregulation for stroke are discussed further in the following section.

3 Physiological Relevance 3.1 General Physiological Roles Ion transport-dependent function. The clinical relevance of the Naþ/Kþ-ATPase is highlighted by the use of cardiac glycoside drugs to increase heart contractility in the treatment of congestive heart failure. Although it is initially counterintuitive that blocking the Naþ/Kþ-ATPase would provide beneficial effects in heart failure, digoxin and other cardiac glycosides are thought to provide this benefit via the mechanism shown in the left panel of Fig. 3. This model centers on the role of the sodium–potassium pump in maintaining ionic gradients within the cell, and provides a general description of the ionic disturbances caused by Naþ/Kþ-ATPase inhibition. In the heart, inhibition of the Naþ/Kþ-ATPase leads to the accumulation of intracellular sodium, which decreases the steepness of the sodium gradient across the membranes of cardiac muscle cells. This reduced sodium gradient in turn limits the activity of the sodium–calcium exchanger, which normally taps the sodium gradient for energy in the extrusion of calcium. Each cardiac action potential is thus followed by elevated levels of residual intracellular calcium, the net effect of which is to strengthen successive heart contractions [89]. In this way, inhibition of the Naþ/Kþ-ATPase by cardiac glycoside drugs, notably digitoxin (Crystodigin) and digoxin (Lanoxin), produces beneficial effects in patients with congestive heart failure. Ion transport-independent signaling. There is increasing evidence that Naþ/ þ K -ATPase can also influence cellular processes by acting as part of a stable signalosome that regulates intracellular signaling pathways [90, 91]. A significant proportion of the sodium–potassium pumps expressed on the surface of certain cell types may not actively pump ions; rather, they may act as receptors for molecules that bind to the pump [92]. Such a signaling function would be thus independent of ionic transport activity and would work through the


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Fig. 3 Effects of pump blockade on ionic homeostasis and intracellular signaling cascades. Top panel: Under normal conditions, each cycle of the sodium–potassium pump transports three sodium ions out of and two potassium ions into the cell. This active, ATP-dependent process creates and maintains the normal sodium gradient across the cell plasma membrane, and secondly powers the extrusion of intracellular calcium via the sodium–calcium exchanger, which couples the inward flow of sodium down its concentration gradient with the outward transport of calcium. Left panel: Inhibition of the sodium–potassium pump degrades the sodium and potassium gradients, allowing a buildup of extracellular potassium and intracellular sodium. The increase in intracellular sodium reduces the sodium gradient that is the driving force for the sodium–calcium exchanger, thus limiting its capacity to extrude intracellular calcium. Right panel: Blockade of the sodium–potassium pump with low doses of ouabain also activates ion transport-independent signaling pathways. Src directly interacts with the pump to activate multiple signaling cascades. The downstream effects of these cascades include activation of the NFkB and AP1 transcription factors, and such signaling through the Naþ/Kþ-ATPase has been implicated in growth, proliferation, apoptosis, and ischemic preconditioning.

activation of second messenger cascades [93]. Low concentrations of pumpblocking compounds like ouabain activate these receptors in an isoformspecific manner because the binding affinities of the a2 and a3 isoforms for ouabain are much higher than that of the a1 and low doses of ouabain specifically inhibit just a subset of the a isoforms [94]. A simplified schematic of signaling downstream of the sodium–potassium pump is given in the right panel of Fig. 3. The binding of pump-blocking molecules like endogenous cardiosteroids or low doses of ouabain activates Src. Src directly interacts with the Naþ/Kþ-ATPase, and the activation of this molecule initiates a cascade involving a number of other signaling

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intermediates, culminating in the downstream activation of the MAPK cascade, PLC, and PKC. While the entire complement of cellular changes resulting from Naþ/Kþ-ATPase signaling remains unknown, signaling downstream of the Naþ/Kþ-ATPase initiates a host of cellular events including alteration of a subunit localization in the plasma membrane in an isoform-specific manner [95]. In turn, the combination of dysregulated intracellular calcium and Naþ/KþATPase signalosome activity induced by pump blockade influences cellular transcription via activation of transcription factors like NFkB and AP-1. Such ion transport-independent Naþ/Kþ-ATPase signaling has been shown to influence physiologically relevant processes such as hypertrophic growth in cardiac myocytes [96, 97], proliferation in smooth muscle [98], and apoptosis in malignant cells [99, 100]. Finally, it is relevant for the present discussion of stroke that MAP kinase activation downstream of Naþ/Kþ-ATPase signaling mediates a form of neuronal pre-ischemic conditioning that protects cells later exposed to hypoxic conditions [101].

3.2 Pathological Relevance to Stroke Ischemic stroke, which is defined by the loss of blood and oxygen flow to a region in the brain, profoundly affects the activity of the sodium–potassium pump. In rat models, ischemia causes ATP levels to drop precipitously within minutes due to the inhibition of mitochondrial respiration [102]. Because the Naþ/Kþ-ATPase uses such a large proportion of the cell’s energy store, its activity becomes especially vulnerable under these conditions. Both Naþ/Kþ-ATPase levels and activity are downregulated in model systems of focal ischemia and traumatic brain injury [103, 104, 105, 106], and the reduction of Naþ/Kþ-ATPase activity is partly responsible for the increased sodium influx observed in the hippocampus during and after anoxia [107]. Naþ/Kþ-ATPase activity is also downregulated in a variety of other neuropathologic and apoptotic contexts clearly associated with compromised cellular health [108, 109, 110]. Surprisingly, however, evidence also suggests that inhibition of the Naþ/KþATPase can be neuroprotective. Low concentrations of ouabain that preferentially inhibit the a2 and a3, but not a1, isoforms are protective against hypoxiainduced cell death in cortical [111], striatal [112], and cerebellar [113] neurons. Such inhibition of the Naþ/Kþ-ATPase promotes neuron survival as well as the activation of factors like PI3K and pAkt that are important for somal health and neurite extension [114, 115, 116, 117, 118]. Independently, in a blinded screen of several thousand known drug-like compounds (including all FDA-approved drugs), we identified a cardiac glycoside as one of the two most efficacious compounds in providing neuroprotection in a cortical brain-slice explant model for focal ischemia (Fig. 4) [119]. This compound, neriifolin, is structurally closely related to the FDA-approved cardiac glycosides digoxin and digitoxin, and indeed these drugs were


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Fig. 4 The cardiac glycoside neriifolin is neuroprotective in models of focal ischemia. a A highthroughput biology screen was developed for ischemic stroke. In this screen, biolistically transfected cortical brain-slice explants were subjected to transient oxygen-glucose deprivation (OGD), causing the degeneration of YFP-transfected neurons over the course of 1–3 days (compare control brain slice (left) to OGD brain slice (right) at 3 days post-OGD). b This brain-slice model for ischemic stroke resulted in the identification of the cardiac glycoside neriifolin as a potent neuroprotective agent. c Summary data from a portion of this screen are shown; the solid line at a value of 1 on the y-axis shows the OGD negative control level whereas the solid line at a value of 28 shows the positive control level for parallel brain slices co-transfected with the antiapoptotic gene Bcl-xL. The dotted line depicts 5 SD above the negative OGD control. While several compounds rescued against OGD-induced neuronal death above this threshold level, neriifolin afforded the greatest neuroprotection in this run (red arrow). d Neriifolin continued to provide neuroprotection even when administered 6 or more hours after OGD. e Neriifolin provides a dose-dependent reduction of infarct volume in the adult rat middle cerebral artery occlusion (MCAO) model of focal ischemia. *P < 0.07, **P < 0.01. Data adapted from [119].

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subsequently also shown to provide neuroprotection in the brain-slice stroke model but with lower potency. Importantly, we found that neriifolin could provide quantitative neuroprotection even when administered 6 or more hours after ischemic injury, corresponding to the therapeutic time window in which stroke victims are first likely to reach the emergency room [120]. Finally, the neuroprotective action of neriifolin was confirmed in two independent in vivo stroke models. It is possible to reconcile these apparently conflicting sets of data by considering the hypothesis that ATP conservation during ischemic conditions can serve to ameliorate cellular damage and/death consequent to mitochondrial collapse. Reducing demand for ATP by inhibiting the brain’s greatest consumer of ATP may thus be able to promote neuronal survival even under adverse conditions such as ischemia. Such a reduction of metabolic demand under conditions of stress is not a novel concept and is consistent with the ‘‘channel lock’’ hypothesis proposed by Hochachka in the 1980s [121]. In fact, reduction in brain energy consumption forms the underpinnings of several adaptive neuroprotective strategies found in the animal kingdom: gall flies survive the a-energetic conditions of the winter hibernation period by reducing Naþ/Kþ-ATPase activity [122]; snails entering a hypometabolic state termed estivation reduce Naþ/ Kþ-ATPase ATP consumption through phosphorylation of the a subunit [123]; sea turtles reduce energy consumption by the Naþ/Kþ-ATPase to survive prolonged underwater hypoxia [124]; ground squirrels show phosphorylation-induced reductions in Naþ/Kþ-ATPase activity during winter hibernation [125]. In addition, that hypoxia in rats stimulates a large and rapid increase in the production of an endogenous ouabain analog to reduce Naþ/Kþ-ATPase activity is another example of adaptive ATP conservation under hypoxic conditions [126]. Finally, a principal mechanism underlying therapeutic hypothermia used in the clinic is the conservation of ATP, much of which would otherwise be used to power the Naþ/Kþ-ATPase [127]. In addition to ATP conservation, blockade of the sodium–potassium pump may also provide neuroprotection in ischemia through modulating intracellular calcium levels just as the cardiac glycosides do in the heart contraction cycle. Although high levels of calcium are clearly harmful for neuronal viability [100], abnormally low levels of calcium can also lead to neuronal death [128, 129]. The ‘‘calcium set-point’’ hypothesis for neuronal trophic dependence thus suggests that elevation of cytosolic calcium is critical for neuronal survival when normal trophic support is not available – as in the case of ischemic stroke. Consistent with this hypothesis, hippocampal neurons exposed to transient ischemia experience lower than normal levels of intracellular calcium and NMDA receptors for 1–3 days subsequent to the ischemic insult [130], and the administration of NMDA receptor agonists to neurons during this period paradoxically attenuates neurologic deficits and restores cognitive performance [131]. Calcium starvation and its consequent apoptosis may even be the predominant cause of cell death in the ischemic penumbra, particularly at later time intervals


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after stroke [132]. Therefore, elevating calcium levels in neurons exposed to ischemic events via suppression of sodium–calcium exchange as a secondary consequence of Naþ/Kþ-ATPase inhibition may be an important mechanism for providing protection against the delayed neuronal death following stroke. In further support of Naþ/Kþ-ATPase inhibition providing neuroprotection to mammalian neurons under stress, low doses of ouabain have been found to increase the survival of both cultured retinal ganglion cells and mesencephalic dopaminergic neurons [133, 134], and a non-cardiac glycoside compound with Naþ/Kþ-ATPase inhibitory properties has recently been found also to provide neuroprotection in an in vivo stroke model [135]. Finally, a screen of the NINDS Custom Collection for new drug targets and candidates for the treatment of spinobulbar muscular atrophy showed that cardiac glycosides inhibit the polyglutamine-dependent activation of caspase-3 and thus provided neuroprotection against polyglutamine-mediated cytotoxicity [136].

3.3 Therapeutic Perspectives Inhibition of the Naþ/Kþ-ATPase thus offers novel neuroprotective mechanisms for providing therapeutic benefit after stroke. This exciting possibility, together with the availability of FDA-approved Naþ/Kþ-ATPase inhibiting drugs, however, is tempered by some points of potential concern. First, the cardiac glycoside drug digoxin, which is in current clinical use, has limited access to the CNS under normal conditions. Although numerous examples of CNS-related side effects of digitoxin treatment argues for some degree of CNS penetration [reviewed in 137], penetration of digoxin into the CNS is limited by the efflux transporter P-glycoprotein in the blood–brain barrier (BBB) endothelium [138]. Targeted deletion of the mdr1a gene in mice lacking this transporter results in substantially higher levels of digoxin penetration into the brain [139, 140], and inhibitors of the transporter (e.g., quinidine and Pluronic p85) also increase digoxin penetration across the BBB [141, 142, 143, 144]. The development of tractable drugs inhibiting the P-glycoprotein transporter therefore may offer a pathway for increasing the penetration of drugs blocking the sodium–potassium pump into the CNS [145]. A second concern over the therapeutic use of Naþ/Kþ-ATPase inhibitors in stroke centers on a problem common to all drugs intended as stroke treatments. Because the average stroke patient does not arrive at the hospital until 6 or more hours after stroke onset [120], any drugs developed for the treatment of stroke must retain their therapeutic effects even after a significant delay of administration. In fact, the beneficial effects of the ‘‘clotbuster’’ tissue plasminogen activator (tPA), the only drug treatment currently FDA-approved for stroke, decline rapidly in the hours after stroke onset and tPA is not indicated for use after 3-h post-stroke onset. This hurdle has produced significant problems for the development of effective therapeutic agents for this disease, and one

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promising aspect of our studies of neriifolin described above is that it retains neuroprotective effects even 6 h or more after ischemic injury in the context of brain tissue explants. Finally, the narrow therapeutic window with respect to dose for cardiac glycosides and other Naþ/Kþ-ATPase inhibitors presents a challenge for clinical management. The beneficial effects of energy conservation due to low levels of Naþ/Kþ-ATPase inhibition could quickly become detrimental in terms of excessive rundown of cellular ionic gradients, particularly in off-target organs such as the heart. In this context, it will be critical, as described above, to develop strategies for increasing the penetration of Naþ/Kþ-ATPase inhibitors into the CNS and ideally to develop such inhibitors with preferential action on neural tissues.

4 Summary As the brain’s main consumer of ATP, the Naþ/Kþ-ATPase is a critical determinant of normal neuronal function in the brain. In addition to its direct role in ionic transport, the sodium–potassium pump is crucial for maintaining key cellular properties (e.g., osmolarity, pH, and membrane potential), modulating important signaling cascades, and controlling intracellular calcium levels. Each of these functions is threatened when Naþ/Kþ-ATPase activity slows in ischemia, but recent evidence from a range of studies suggests that pharmacological inhibition of the pump’s energy requirements may in fact promote neuron survival under ischemic conditions. Based on this evidence, the Naþ/KþATPase clearly merits additional study as a potential drug target for ischemic stroke.

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Acid-Sensing Ion Channels (ASICs): New Targets in Stroke Treatment Giuseppe Pignataro

1 Introduction In the last decades enormous effort has been made in an attempt to attenuate stroke injury. Despite the attractiveness of the excitotoxicity hypothesis, all clinical trials using glutamate antagonists as protective agents against ischemia have failed. This may be partially due to other injury pathways and non-excitotoxic processes involved in the complex mechanisms triggered by the lack of blood supply in brain. In this regard, the role played by acid-sensing ion channels (ASICs) in cerebral ischemia has been recently underlined [15, 57, 83]. A stable pH is critical for normal cellular function [24]. In physiological conditions, extracellular pH (pHo) and intracellular pH (pHi) are maintained at 7.3 and 7.0 through different Hþ-transporting mechanisms [24]. In pathological conditions such as tissue inflammation, neurotrauma, epileptic seizure, and especially ischemic stroke, accumulation of lactic acid due to enhanced anaerobic glucose metabolism and release of Hþ from ATP hydrolysis result in marked reduction of tissue pH, a condition termed acidosis. In particular, during severe ischemia brain pH can drop to as low as 6.0 [25, 57, 62]. It has been known for several decades that acidosis occurring after ischemia is associated with neuronal injury [67]. Recent studies have reported that activation of the Ca2þ-permeable ASIC1a plays a pivotal role in the development of acidosis-mediated ischemic damage [57, 83, 86]. These findings dramatically changed the view of acid signaling and offered new pharmacological targets for those

G. Pignataro (*) Department of Neuroscience, School of Medicine, Federico II University of Naples, Via Sergio Pansini 5, 80131 Naples, Italy e-mail: [email protected]


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neurological diseases in which acidosis is a relevant pathophysiological event [15, 57, 79, 83, 86].

2 Biochemical and Molecular Biology of the Plasma Membrane Protein Since the first subunit was cloned in 1997 [75], six ASICs, encoded by four genes, have been identified: ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, and ASIC4. All ASICs belong to the degenerin/epithelial Naþ channel (DEG/ ENaC) superfamily, which are Naþ-selective cation channels sensitive to the Kþ-sparing diuretic amiloride [3, 75]. ASICs are found throughout the mammalian central and peripheral nervous systems with ASIC1a, ASIC2a, and ASIC2b present in the brain, especially in cerebral cortex, cerebellum, hippocampus, amygdala, and olfactory bulb, and ASIC1a, ASIC1b, ASIC2a, ASIC2b, and ASIC3, abundant in the sensory neurons of the peripheral nervous system [4, 75, 77]. Based on the biochemical analysis of ENaC [63, 75] and the glycosylation studies of ASIC2a subunit [65], the proposed membrane topology of each ASIC subunit consists of two transmembrane domains (TM1 and TM2), linked by a large extracellular cystein-rich loop, and intracellular N- and C- termini. Functional ASICs are believed to be tetrameric assemblies of homomeric or heteromeric subunits [50]. However, based on the stoichiometric studies of other DEG/ENaC, the possibility that ASICs are assembled with three to nine subunits cannot be excluded [35, 38]. Recently, with the aid of crystallographic studies it has been possible to state that ASIC1 subunit stoichiometry is trimeric, and thus the obligate heteromeric ENaCs are almost certainly trimers, defined by an A1B1C1 stoichiometry [47]. Furthermore, the large extracellular portion of ASIC1 polypeptide is composed of multiple domains that together form a complex structure rich in cavities and protrusions and perhaps prominent ‘‘handholds’’ for additional interacting proteins. Multiple close acidic residue pairs have been identified as proton-binding sites, thus demonstrating that the pH-sensing sites are distributed in primary structure and 3D space, far from the channel region. To explain how proton binding at these distant sites is translated into channel gating, it has been suggested that the disulphide-rich thumb domain undergoes movement on proton binding/unbinding and that these movements are transmitted to the ion channel transmembrane domain by way of primarily non-covalent interactions, mediated by residues at the base of the thumb. Trp 288 (thumb) may act like a ball sitting in a socket-like joint of the transmembrane domain and perhaps when the thumb domain moves, its movement is coupled to the transmembrane domain by way of this thumb–transmembrane domain ball-and-socket joint (Fig. 1) [47].

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Fig. 1 Putative structure of acid-sensing ion channel 1(ASIC1)

3 Tissue Distribution In the CNS, ASIC1 is the most abundantly expressed channel of the ASIC family [41, 75]. With the aid of in situ hybridization, ASIC1 mRNA has been found in the olfactory bulb, cerebral cortex, hippocampus, basolateral amydgaloid nuclei, subthalamic nuclei, and cerebellum [41, 75]. It should be here underlined that effective activation of ASIC1 requires rapid and large decreases in pHo of 1 pH unit [4, 10, 14, 75]. Protons also induce desensitization, thus, re-activation of channels requires the pHo to return to values >7.3. Recovery from desensitization is slow, with > Ba2+ > Co2+ > Mg2+ > Mn2+ > Sr2+ > Cd2+ > Ca2+ [27]. Though the resulting currents are admittedly small or even un-resolvable, divalent entry can be observed in experiments using fluorescent dyes. In this way, Monteilh-Zoller et al [27] elegantly demonstrated that TRPM7 allows entry of divalents, including Ca2+, Mn2+, Co2+, and Ni2+, despite the presence of physiological levels of extracellular Ca2+ and Mg2+. Admittedly, neurons possess numerous influx pathways for trace metal ions, most notably voltage-gated Ca2+ and NMDAR channels. However, unlike 2+

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these channels, which are normally quiescent until activated (i.e., by voltage or by agonist), TRPM7 is constitutively active. Consequently, though modulated by several extracellular and intracellular factors (e.g., ROS/RNS, pH, and intracellular Mg2+, described in the following section), the current flowing through these channels at any given time is predominantly limited by the extracellular concentration of extracellular divalents. It is likely this feature, which allows TRPM7 to play an important part in maintaining intracellular Mg2+ homeostasis and also to ‘‘sense’’ changes in the extracellular concentration of divalents [25]. Such changes occur during intense neuronal activity associated, for example, with seizure and especially ischemic episodes.

7 Regulatory Mechanisms: Receptorial, Transcriptional, and Transductional In addition to regulating channel activity from the extracellular space, Mg2+ plays an important role in regulating TRPM7 intracellularly. Previous studies have clearly demonstrated that intracellular Mg2+ inhibits TRPM7 channels. Consequently, experimental conditions which reduce the intracellular concentration of this divalent cation (e.g., intracellularly applied Mg2+ chelators such as HEDTA or Na-ATP) facilitate TRPM7-mediated currents. More controversial in whether or not the intracellular block is mediated solely by Mg2+ or rather by Mg2+ nucleotides [12, 28]. Studies in which the kinase activity of the channel was modified (or entirely removed) by mutation suggest that nucleotide binding to this domain may act as a gating modifier by altering the sensitivity of the channel to inhibition by Mg2+ at a distinct site. The demonstration of an interaction between PLC-b1 and TRPM7 led Clapham’s group to explore the functional consequence of this interaction. They demonstrated that the interaction allows the concentration of phosphatidylinositol 4,5-bisphosphate (PIP2) to be regulated in the vicinity of the channel. In this way, Gaq-linked G-protein receptors that activate PLC inhibit TRPM7 through the hydrolysis of PIP2 [29]. However, a recent report [30] proposes a more complex regulation by PLC-coupled receptors. Using less invasive perforated-patch recordings, in cells expressing TRPM7 at modest levels, the authors reported that PLC-coupled receptor agonists can enhance rather than inhibit TRPM7 function. They suggest that the resting intracellular concentration of Mg2+, which may be influenced by the recording configuration used (i.e., perforated patch versus whole-cell recording techniques), may determine the outcome of PLC stimulation. TRPM7 may also be regulated through phosphorylation. Interestingly, the closely related TRPM family TRPM6, which may form heteromeric channels with TRPM7, can phosphorylate TRPM7 at threonine residues [31]. A recent study identified a variant of TRPM7 in a subset of Guamanian amyotrophic lateral sclerosis and parkinsonism dementia patients [32]. The variant harbored a missense mutation (T1482I) associated with reduced phosphorylation and

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increased sensitivity to inhibition by Mg2+. Though the specific sites have yet to be identified, these studies suggest that phosphorylation by TRPM6 could conceivably play an important role in regulating TRPM7 by altering the channel sensitivity to Mg2+.

8 Physiological Properties To date, most of the available information for the physiological functions of mammalian TRPM7 channel is drawn from cellular studies. There are unconfirmed reports that TRPM7 knockout is embryonically lethal. At the cellular level, TRPM7 overexpression in heterologous cell systems (e.g., HEK-293) has a detrimental effect on cell survival [5]. Normally adherent HEK-293 cells detach and ultimately die off upon TRPM7 overexpression [5]. Two other studies [33, 34] also confirmed that TRPM7 overexpression disrupts cell adhesion. Both studies indicated the involvement of Ca2+-dependent mechanisms. Paradoxically, TRPM7 knockout in avian B cells caused cell death indicating its vital role in cell survival [11]. TRPM7 appears to have vital role in Mg2+ homeostasis in DT-40 cells as high Mg2+-containing medium, but not high Ca2+, can restore normal cell growth and proliferation in TRPM7-deficient cells. The diffuse expression of TRPM7 suggests that it may function as a regulator for cellular Mg2+ homeostasis in numerous physiological systems [35, 36]. In addition to Mg2+, the TRPM7 channels contribute to Ca2+ influx into cells and thereby may regulate cell proliferation and G1/S cell cycle progression [24]. Similarly, TRPM7 orthologs have been identified in non-mammalian systems such as zebra fish and Caenorhabditis elegans [37] and knockdown of these channels affects cell growth and proliferation [38]. Also, in C. elegans, the TRPM7 ortholog has been implicated as a component of the defecation rhythm generator [39]. A recent study suggests a similar role for TRPM7 in mammals [23].

9 Pathophysiological Relevance in Stroke Numerous clinical trials of AETs have failed to benefit stroke patients [40]. To date, there is still no clinical or experimental treatment that can improve the outcome of stroke patients, with the exception of the thrombolytic agent, tissue plasminogen activator (tPA). This remains true despite significant advances in our understanding of ischemic mechanisms over the last two decades. A few years ago, we described two key mechanisms that cause neuronal injury when neurons are challenged by anoxia or excitotoxins. The first mechanism involves the activation of signaling pathways associated with NMDA glutamate receptors (NMDARs) through submembrane scaffolding proteins such as PSD-95 [41–43]. The second mechanism is through the activation of TRPM7 channels [25, 44]. The Ca2+ influx contributed by these channels is a major contributor to

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the neuronal damage observed in hypoxic and ischemic conditions. Since TRPM7 is widely expressed in different tissues and systems including brain, heart, kidney, and blood vessels it is likely that these channels may play an important role not only in cerebral ischemia but also in ischemic injury in the heart and kidney, for example. Our group [44] demonstrated a role for TRPM7 in neurons in an experimental model of ischemia. TRPM7 was shown to contribute to hypoxic neuronal death in rodent cultured cortical neurons subjected to prolong oxygen and glucose deprivation (OGD). In this model, Ca2+ influx through TRPM7 channels was correlated with neuronal cell death and suppression of TRPM7 expression, using siRNA, inhibited an OGD-evoked current and protected neurons against OGD-induced neuronal death. Interestingly, in the context of the recruitment of TRPM7 during ischemia, TRPM7 can be positively modulated by both protons [45] and by ROS/RNS [44]. As a result, the decrease in extracellular divalents, increase in extracellular proton concentration and the generation of ROS/RNS that occur coincidently during ischemia will act in concert to powerfully recruit TRPM7 channels (Fig. 4).

Fig. 4 Model of TRPM7 activation during cerebral ischemia. Strong NMDA receptor activation during the initial stages of an ischemic attack leads to a large influx of Ca2+. The resulting Ca2+-dependent stimulation of NOS, tethered to NMDARs through PSD-95, and O2– production from mitochondria leads to the production of ONOO–, which stimulates TRPM7 channels. The reduction of extracellular divalents and decrease in pH, both associated with ischemic episodes, further enhance TRPM7 activation. These factors act in concert to stimulate additional Ca2+ influx through TRPM7 channels leading to a potentially toxic positive feedback


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In addition to contributing to the Ca2+ loading of neurons, the permeability of TRPM7 to toxic metal ions (e.g., Zn2+, Ni+2, and Cd2+) may also play a role. In particular, Zn2+ is highly toxic to cells if its concentration exceeds the trace level. In this way, the Zn2+ permeability of TRPM7 may contribute not only to acute neurotoxicity in ischemia [46, 47] but may also be a factor in the pathogenesis of chronic neurodegenerative conditions like Alzheimer’s disease where Zn2+ has been reported to accumulate in amyloid plaques [48]. Despite compelling in vitro evidence, the role of TRPM7 in stroke will need to be clarified in in vivo studies. Indeed, even though the molecular and cellular illustrations outlined above are crucial, hypoxia or OGD models are no substitutes for ischemic models in whole animals. Events associated with ischemic episodes in vivo may not be faithfully paralleled in the in vitro hypoxic setting [49]. Correspondingly, there is a need to study animal ischemia models to confirm the role of TRPM7 in stroke.

10 Pharmacological Modulation To date, there are no selective agonists and antagonists for TRPM7 channels. Accordingly, we and others have used gene silencing by small interfering RNA (siRNA) to knock down TRPM7 in CNS neurons [25, 44], PNS neurons [26], vascular smooth muscle cells [50], interstitial pacemaker cells of Cajal in the gastrointestinal tract [23], and human epithelial cells [51]. Despite the usefulness of these powerful approaches, there is a pressing need for the development of specific pharmacological agents for studying TRPM7 function.

11 Preliminary Clinical Trials As there are no studies of TRPM7 function in vivo and no specific pharmacological blockers available, we do not expect to see any preliminary clinical trials in the near future. Until drug screening efforts for TRPM7 blockers are successful, siRNA-induced gene silencing may be a potential approach for stroke treatment, as gene therapy has been utilized in other systems [52]. Other approaches aimed at modulating TRPM7 by interrupting specific signaling pathways, as we did in interrupting the NMDA receptors and downstream signal pathway PSD95 [41, 42], may also prove useful.

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12 Therapeutic Perspectives In animal studies, the most effective way to reduce stroke damage is by interrupting the injury process before any damage becomes irreversible. To date the most promising strategies for stroke treatment involves attempts either to improve perfusion using thrombolytic agents (such as tPA) or to limit the injury area using so-called neuroprotective agents. Using tPA is likely able to reduce damage to neurons by re-establishing the blood flow to the ischemic region and this drug is the only treatment currently available for treatment of acute cerebral stroke [53]. Approved by the FDA in 1996, tPA was initially developed for the treatment of heart attack. In spite of being the only approved drug therapy for stroke, less than 1% of stroke patients benefit from tPA because of its limited 3-h therapeutic window (if applied by i.v. administration). Treatment within 90 min of the onset of cerebral stroke maximizes the potential benefit and minimizes the potential risks of tPA. The most dangerous risk is of excess cerebral bleeding from the inappropriate use of tPA, which further complicates the problem of cerebral ischemia. There are also many other limitations for tPA treatment: (1) only 5% of stroke patients are able to make it to a hospital within the 3h therapeutic window; (2) a brain scan must be performed to rule out any sign of intracranial cerebral bleeding; (3) there is an increased risk of intracerebral hemorrhage; and (4) there is a lack of trained personnel available to perform the pretreatment assessment together with tPA administration. In the end, only 1–2% of stroke patients are treated with tPA and, of those, only 30–35% show any beneficial effect of the tPA treatment. Thus, it is of great importance to identify effective neuroprotective medicines and therapeutic measures in order to protect the brain and improve recovery in patients suffering a stroke. In the future, TRPM7 channel modulators might be used in combination therapy for the treatment of cerebral ischemic stroke. Combination therapy for ischemic stroke would ideally help restore cerebral blood flow, while selected pharmacological agents, targeting select targets and applied at empirically determined time points of stroke onset, would provide neuroprotection and potentially facilitate recovery of function (Fig. 5). One potential caveat is that the widespread expression of TRPM7 may limit the selectivity of pharmacological agents developed to target this channel. Since reductions in extracellular divalents are associated with ischemic episodes, it may be possible to improve upon the selectivity by developing activity-dependent blockers that would preferentially target TRPM7 channels strongly recruited during stroke.


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Fig. 5 Ischemic cell death cascade. The recruitment of NMDARs is central to the excitotoxic model of ischemic cell death. The failure of NMDAR antagonists to provide protection in clinical stroke trials has forced a re-evaluation of this model. We propose that the contribution of NMDARs, though critical as an early trigger of death cascades, is limited in time to a brief period immediately following a stroke. Our hypothesis is that the progressive recruitment of additional non-selective cation channels plays an equal, if not greater role, in mediating neuronal death. Rational polytherapy combining drugs that target various contributors of neuronal death may prove to be the most effective means of providing neuroprotection for stroke patients

Conclusions Knockdown of TRPM7 in vitro using siRNA gene silencing has been shown to provide neuroprotection in in vitro models pointing toward an alternative therapeutic approach to stroke treatment. Future studies will likely proceed via a rational polytherapy for stroke treatment involving pharmacological agents that target different ion channels, including TRPM7, receptors and exchangers, as well as acting to restore cerebral blood flow.

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Subtypes of Voltage-Gated Ca2þ Channels and Ischemic Brain Injury Soon-Tae Lee, Daejong Jeon, and Kon Chu

1 Introduction Intracellular Ca2þ ([Ca2þ]i) initiates physiologic events as diverse as gene transcription, muscle contraction, cell division, and exocytosis. Disruption of Ca2þ regulation in the brain accompanies numerous neurological dysfunctions, including epilepsy and ischemia. Although the metabolic machinery that elicits changes in intracellular Ca2þ concentration is heterogenous, voltage-gated calcium channels (VGCCs) are one of the most important components of neurons and other excitable cells, and they play major roles in both normal functioning and various pathological processes, such as cerebral ischemia, epilepsy, migraine, muscle weakness, and cerebellar ataxia. VGCCs are a group of voltage-gated ion channels found in excitable cells with permeability to the Ca2þ ion. Since VGCCs were first identified in crustacean muscle in 1953 by Paul Fatt and Bernard Katz [29], researchers have identified several different kinds of VGCCs, including L-, N-, P/Q-, R-, and T-type calcium channels. VGCCs are normally closed at physiologic or resting membrane potential. When they are activated (i.e., opened) at depolarized membrane potentials, VGCCs allow Ca2þ to enter the cell, resulting in muscular contraction, excitation of neurons, upregulation of gene expression, activation of Ca2þ-dependent enzyme, neurite growth, or release of neurotransmitters. Given their importance in cerebral ischemia, this review describes the structures, functions, phenotypes of knockout mice, and pathophysiologic relevance of VGCCs in cerebral ischemia.

K. Chu (*) Department of Neurology, Seoul National University Hospital, 101, Daehangno, Jongno-Gu, Seoul, 110-744, South Korea e-mail: [email protected]


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2 Structure and Subunits of VGCCs VGCCs have multi-subunit structures consisting of a major pore-forming subunit (a1) and several auxiliary subunits (a2–d1–4, b1–4, and g1–8) (Fig. 1). The purification of VGCCs was started from 1,4-dihydropyridines (DHPs)-binding Ca2þ channels, which are highly expressed in T-tubules [15, 52]. The a1 protein was identified as the component that bound to DHPs. Cloning of the cDNA for the DHP-binding channels was accomplished initially from skeletal muscle [121], and subsequently from heart, by homology with the skeletal muscle sequence [80]. The general structure of a calcium channel is biochemically complex and composed of four or five distinct subunits [15]. The various VGCCs are structurally homologous, but not identical. The a1 subunit is the largest subunit (190–250 kDa), and the a1 subunit comprises four homologous (or conserved) domains (I–IV) that are linked by variable cytoplasmic sequences (intracellular connecting loop). Each homologous domain (I–IV) contains six transmembrane a-helix segments (S1–S6), and the S4 segment is the voltage sensor (Fig. 1). The a1 subunit harbors the molecular machinery to produce a functional channel, such as p-loops that permit ion permeation and selectivity, voltage sensors that allow the channel to respond to membrane depolarization, intrinsic inactivation machinery, and the binding sites for all known Ca2þ channel blockers. The pore of VGCCs is

Fig. 1 Structural organization of VGCCs. VGCCs have multi-subunit structures consisting of a major pore-forming subunit (a1) and several auxiliary subunits (a2–d, b, and g) [107]

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exquisitely Ca2þ selective, and this property has been attributed to key negatively charged residues, usually glutamate, in each pore loop [26, 131]. Thus, the a1 subunit pore is the primary subunit required for channel functioning. Most Ca2þ channels contain an intracellular b subunit and a transmembrane, disulfide-linked a2d subunit complex. Among the auxiliary subunits, the b subunits are entirely cytosolic (Fig. 1). Four b subunits (Cavb1–4) have been cloned and each Cavb has distinctive properties [25]. Structurally, Cavb has five different domains, with the two conserved domains sharing significant homology among b subunits. The conserved domains were revealed as a src homology-3 (SH3) domain and a guanylate kinase (GK) domain [18, 41, 90, 125], and thus Cavb is included in membrane-associated guanylate kinase (MAGUK) family that has scaffolding functions. Interestingly, recent several reports suggest that Cavb can bind to other molecules and work without marked influence on VGCCs [6, 8, 32, 39, 46, 47, 59, 99]. Cavb has marked effects on the properties of VGCCs a1 subunits, including trafficking of Ca2þ channel complexes to the plasma membrane, voltage-dependence, and activation/inactivation kinetics of Ca2þ currents [9, 25]. The a2 and d auxiliary subunits are encoded by a single gene, and their polypeptide is post-translationally cleaved into an a2 and a d (Fig. 2). The post-translationally cleaved products form disulfide-linked a2–d. a2 is entirely extracellular and d has a single transmembrane domain, which serves to anchor a2d complex in the plasma membrane. a2 is extensively glycosylated, and this post-translational modification gives the stability of the interaction with a1. Four a2d subunit genes have now been cloned. a2d subunit was also shown to modify the biophysical and pharmacological properties of the a1 subunit [4, 68]. Eight g subunit has four transmembrane domains and eight g subunit genes have now been identified (Fig. 1). The g1 subunit is known to be associated with skeletal muscle VGCC complexes, and other g subunits are expressed in the

Fig. 2 The phylogenetic tree illustrates the amino acid sequence homology of various VGCCs (modified from [28])

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heart and brain [16]. The pharmacological and electrophysiological diversity of VGCCs arises primarily from the presence of multiple a1 subunits, and the remaining auxiliary subunits (b, a2d, g) modify the properties of the channel complex [51].

3 Classification of VGCCs The a1 subunits are encoded by at least 10 distinct genes, called CACNA1 (voltage-gated calcium channel a1). The Ca2þ channel genes were named using the chemical symbol of the principal permeating ion (Ca) with ‘‘voltage’’ indicated as a subscript (CaV) [28]. The numerical identifier corresponds to the CaV channel a1 subunit gene subfamily (1–3) and the order in which the a1 subunit was discovered within its respective subfamily (1–n). According to this nomenclature, L-type Ca2þ currents are mediated by the CaV1 subfamily (CaV1.1–CaV1.4), which includes channels containing CACNA1S, CACNA1C, CACNA1D, and CACNA1F (also referred to as a1S, a1C, a1D and a1F, respectively) (Table 1). P/Q-type, N-type, and R-type Ca2þ currents are mediated by the CaV2 subfamily (CaV2.1–CaV2.3), which includes channels containing CACNA1A, CACNA1B, and CACNA1E (referred to as a1A, a1B, a1E, respectively) (Table 1). T-type Ca2þ currents are mediated by the CaV3 subfamily (CaV3.1–CaV3.3), which includes channels containing CACNA1G, CACNA1H, and CACNA1I (referred to as a1G, a1H and a1I, respectively). This classification of VGCCs is based on electrophysiological and pharmacological properties [15, 28, 52] (Tables 1 and 2). The amino acid sequences of the a1 subunits within a subfamily share more than 70% identity, but they share less than 40% identity among the three subfamilies [16] (Fig. 2). The genes for the various a1 subunits have become widely dispersed in the genome and are not clustered on single chromosomes in mammals.

4 Biophysical and Electrophysiological Properties Researchers classified two current components (low- and high-voltageactivated current) in mammalian sensory neurons according to their biophysical properties, showing different conductances in the single channels [14, 30] (Table 2). At first, pharmacological studies using DHPs defined L-type VGCC with ‘long lasting’ conductance as high-voltage activated (HVA) channels present in skeletal muscle, heart, smooth muscle, and neurons [45]. Pharmacological studies further revealed the presence of non-L-type HVA channels [110]. The DHP-insensitive, non-L-type current component was then subdivided according to its biophysical properties [88] and several irreversible toxins [89]. Two subtypes of non-L-type HVA channels were identified. First, N-type (for non-L and neuronal) channels were blocked by a toxin fraction

SNX-482 None

CACNA1D

CACNA1F

CACNA1A

CACNA1B

CACNA1E

CACNA1G CACNA1H CACNA1I

Cav1.3

Cav1.4

Cav2.1

Cav2.2

Cav2.3

Cav3.1 Cav3.2 Cav3.3

P/Q-type

N-type

R-type

T-type

Specific antagonists

o-Conotoxin-GVIA

o-Agatoxin IVA

Dihydropyridines, phenylalkylamines, benzothiazepines

Cav1.1 Cav1.2

L-type

Protein

CACNA1S CACNA1C

Gated by

Current type Skeletal muscle, transverse tubules. Cardiac myocytes, smooth muscles, endocrine cells, neuronal cell bodies, proximal dendrites Endocrine cells, neuronal cell bodies and dendrites, cardiac atrial cells and pacemaker cells, cochlear hair cells Retinal rod and bipolar cells, spinal cord, adrenal gland, mast cells Purkinje neurons and granule cells in the cerebellum, nerve terminals and dendrites, neuroendocrine cells Throughout the brain, nerve terminals and dendrites, neuroendocrine cells Neuronal bodies and dendrites, cerebellar granule cells Neuronal cell bodies and dendrites, cardiac and smooth muscle myocytes

Distribution

Repetitive firing, dendritic calcium transients. Pacemaking, repetitive firing

Neurotransmitter release, dendritic Ca2þ transients, hormone release

Excitation-contraction coupling Excitation-contraction coupling, hormone release, transcriptional regulation, synaptic integration Hormone release, transcriptional regulation, cardiac pacemaking, hearing, neurotransmitter release from sensory cells Neurotransmitter release from photoreceptors Neurotransmitter release, dendritic Ca2þ transients, hormone release

Cellular function

Table 1 Subtypes and physiologic functions of voltage-gated calcium channels

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Table 2 Electrophysiological properties of HVA and LVA VGCCs. Each type of VGCCs has a distinct kinetics and a conductance [48] HVA LVA L P/Q, N, R T Activation range

Positive to –30 mV

Inactivation range Inactivation

–60 to –10 mV Very slow (t > 500 ms) Rapid 25 pS

Deactivation rate Single-channel conductance Single-channel openings Relative conductance Divalent block

Continual reopening Ba2þ > Ca2þ Cd2þ > Ni2þ

Positive to –20 mV –120 to –30 mV Partial (t = 5080 ms) Slow 13 pS Long burst Ba2þ > Ca2þ Cd2þ > Ni2þ

Positive to –70 mV –100 to –60 mV Complete (t = 2050 ms) Rapid 8 pS Brief burst, inactivation Ba2þ = Ca2þ Cd2+ < Ni2+

from the fish-eating cone shell, termed o-conotoxin GVIA. Second, a very slowly inactivating Ca2þ current that was insensitive to both DHP and oconotoxin GVIA was identified in Purkinje neurons of the cerebellum [49]. This current (P-type for Purkinje) was found to be sensitive to a component of the venom (o-agatoxin IVA) from the American funnel web spider, Agelenopsis aperta [19, 81]. Another o-agatoxin IVA-sensitive current showing more rapid inactivation was subsequently identified in cerebellar granule cells. This was originally termed Q-type current [97]. However, it was found that these two currents are elicited by different splicing forms of the same gene, and now these currents are termed as P/Q-type [11, 61]. There was also a residual or R-type Ca2þ current that is resistant to DHPs and the N- and P/Q-type VGCCs toxins [97]. Originally R-type Ca2þ current was included in low-voltage-activated (LVA) channels according to its activation or inactivation voltage ranges. But, now this is included in HVA channels, due to the clear difference from LVA channels. R-type Ca2þ channel is an intermdiate voltage-activated channel and is certainly more inactivating than the other high-votage-activated channels. In comparison of the homology, LVA T-type VGCCs are clearly divergent in terms of structure from the HVA channels [92] (Fig. 2). T-type VGCCs have a tiny conductance and make a transient Ca2þ current. LVA T-type VGCCs also show distinct electrophysiological and pharmacological properties compared to those of HVA VGCCs (Table 2). Neuronal T-type Ca2þ channels (Cav3.1, 3.2, and 3.3) are concentrated on the cell body and dendrites and are important in modulating neuronal excitability. They have several unique properties, including activation at very negative potentials, and the ability to promote burst firing in neurons [57, 77, 112]. T-type VGCCs require strong hyperpolarization to recover from inactivation.


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5 Genetics and Molecular Biology The genetic analysis of VGCCs provides an approach to defining channel function that is complimentary to pharmacological, electrophysiological, and other molecular ways. For an understanding of in vivo functions of VGCCs, mice lacking various a1 subunits were generated in the last few years, and then the functions of VGCCs at the whole animal level have been well described [74, 85, 93]. To study the physiological or pathological roles of L-type Ca2þ channels in vivo, knockout mice were generated. The null homozygous Cav1.2 (a1C) mutant mice died before day 14.5 postcoitum (p.c.) due to the problem of heart development, which suggests that the Cav1.2 VGCC has a critical role in the developmental stage of heart [105]. To overcome this embryonic lethal condition, Cav1.2 mouse was generated using Cre/LoxP site-specific recombination system, which allows time and tissue-specific gene deletion [79, 83, 96]. Cav1.2 was selectively deleted in forebrain including hippocampal formation and cerebral cortex using Nex-Cre donor mice [104]. Forebrain-specific Cav1.2 mutant mice were viable, and showed normal N-methyl-D-aspartate receptor (NMDAR)-dependent long-term potentiation (LTP) in the Schaffer collateral/ CA1 pathway. However, these mice showed the impairment of NMDARindependent LTP. The Cav1.2 mutant mice showed not only reduced late phase of LTP (L-LTP) that requires protein synthesis, but also exhibited impairment in spatial learning in the water maze and labyrinth maze tasks. These results demonstrate the functional relevance of Cav1.2 channel-mediated Ca2þ influx for synaptic plasticity, transcriptional activation, and learning and memory [84]. The Cav1.3 (a1D), another L-type Ca2þ channel, null homozygote mice were generated. These mutant mice were deaf, and both inner and outer hair cell populations were degenerated in the mutant mice by postnatal day 35 and had degeneration of outer and inner hair cells [95]. Notably, however, these mutant mice did not show any effects on LTP in the hippocampus and learning/ memory [23]. Mice lacking Cav2.1 (a1A) exhibited several neurological dysfunctions. Although mutant mice were not embryonically lethal, they showed ataxia and dystonia at about 10 days after birth and could not survive over 4 weeks [33, 61]. In addition, absence seizures, characterized by 3–5 Hz cortical spike-wave discharges (SWDs), which were revealed by electroencephalogram (EEG) recording, associated with behavioral immobility appeared [109]. EEG recordings also showed persistent absence status and a dramatic reduction of gamma-band activity, indicating P/Q-type Ca2þ channels are essential in the generation of gamma-band activity and resultant cognitive function [75]. Disruption of the Ca2þ influx mediated by the gene encoding Cav2.1 altered the expression and functional properties of other Ca2þ channels in neurons. With respect to

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synaptic transmission, Cav2.1 mutant mice displayed impaired excitatory transmission in the hippocampus, but they showed enhanced reliance on Ca2þ influx through N-type or R-type Ca2þ channels in neurons [61]. The Cav2.1 mutant also displayed several alterations in presynaptic function in the neuromuscular junction [123]. The N-type Ca2þ channels are predominantly localized in the nervous system and have been considered to play a vital role in transmitter release at presynaptic terminals, along with P/Q-type Ca2þ channels. The null knockout mice that are genetically deficient in the a1B subunit, unexpectedly, had a normal life span, unlike Cav2.1 mutant mice [44, 56, 64, 100]. However, the functional analysis in vivo revealed several behavioral aberrations and alterations of their nervous system. A large number of studies suggested that the Cav2.2 subunit of N-type calcium channel plays an important role in pain perception at the spinal level, as well as at the supraspinal level. The mutant mice exhibited a significant decrease in anxiety-related behaviors [10, 100, 117]. It was found that the sensitivity to halothane was significantly increased in Cav2.2 mutant mice compared with the wild-type littermates [120], but voluntary ethanol consumption was reduced [86]. With respect to synaptic transmission, the mutant mice exhibited reduced brain-derived neurotrophic factor (BDNF)-induced synaptic potentiation and facilitation. In addition, they showed impaired learning and memory in the Morris water maze and the social transmission of food preference tasks, suggesting that an interaction between N-type VGCCs and BDNF may be involved in the presynaptic enhancement of LTP and normal hippocampus-dependent learning and memory [60]. Otherwise, the role of N-type VGCC in regulation of aggression by modulating inhibitory neurotransmission in dorsal raphe nucleus was revealed [63]. Some compensatory activities were reported in Cav2.2 mutant mice. The mRNA expression of Cav2.1 subunit was increased in cerebellum, olfactory bulb, and adrenal gland [114–116]. The physiological function of R-type Ca2þ channels was not clear, compared with other types of Ca2þ channels. However, Cav2.3 mutant mice gave an advance to understanding of R-type Ca2þ channels. Cav2.3 mutant mice showed reduced spontaneous locomotor activities and increased anxiety level [71, 101]. They also displayed a reduced response to somatic inflammatory pain and an altered response to visceral inflammatory pain, without any difference in acute pain responses [101], suggesting the involvement of R-type Ca2þ channels in controlling the excitability of neurons in the descending antinociceptive pathways (periaqueductal gray matter and/or raphe magnus neurons) [102]. Mutant mice also showed decreased anesthetic sensitivities to propofol and halothane [119]. Acute administration of cocaine normally enhances the locomotor activity in animals, but failed to produce any response in Cav2.3 mutant mice [40]. The studies of synaptic plasticity in Cav2.3 mutant mice revealed that presynaptic Ca2þ entry through R-type Ca2þ channels contributes to the


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induction of mossy fiber LTP, while they did not play a role in fast synaptic transmission [24]. They also showed deficits in long-term depression (LTD) at the parallel fiber–Purkinje cell synapse and motor learning in rotarod tests, suggesting the contribution of Cav2.3 Ca2þ channels to the regulation of activity of Purkinje cells related to motor learning [91]. Mice with a global deletion of the Cav3.1 channels showed absence of the rebound burst-mode potentials in the thalamic neurons, as no difference was observed in tonic-mode firings [66]. Cav3.1 mutant mice were resistant to the genesis of spike and wave discharges induced by baclofen or g-butyrolactone. However, the mutant mice exhibited behavioral seizures in response to systemic administration of bicuculline or 4-aminopyridine. Cav3.1 mutant mice showed hyperalgesia to visceral pain induced by acetic acid [65]. The role of T-type Ca2þ channels in brain rhythms during sleep and their contribution to behavioral states of sleep were revealed [2, 69]. With EEG recordings, Cav3.1 mutant mice showed a loss of delta (1–4 Hz) waves and a reduction of sleep spindles (7–14 Hz) in the thalamus [70]. Furthermore, conditional deletion of Cav3.1 in the rostral-midline thalamus led to frequent and prolonged arousal, which fragmented and reduced sleep in mice. However, sleep was not disturbed in the mice with deletion of Cav3.1 in cortical pyramidal neurons. These findings provide that the Cav3.1 in the thalamus, but not the cortex, is required for stabilized sleep, and this is consistent with a notion that sensory suppression in the thalamus prevents disruption of sleep [2]. Coronary arteries isolated from Cav3.2 mutant arteries showed normal contractile responses, but reduced relaxation in response to acetylcholine and nitroprusside, indicating Ca2þ influx through Cav3.2 Ca2þ channels is essential for normal relaxation of coronary arteries [17]. Cav3.2 mutant mice were used to study pain susceptibility. In acute pain tests, Cav3.2 mutant mice showed decreased pain responses to both acute (mechanical, thermal, and chemical stimuli) and tonic noxious stimuli (intraperitoneal injections of irritant agents and intradermal injections of formalin) [20]. In addition, Cav3.2 mutant mice did not show any responses for GABA-induced cell excitability and intracellular Ca2þ increase in the medium-sized dorsal root ganglia neurons [3]. These results suggest that Cav3.2 T-type Ca2þ channels may be involved in both spinal reflex and supraspinal pain modulation. There was little study of the roles of Cav3.3 T-type Ca2þ channels in the nervous system and Cav3.3 Ca2þ channel-deficient mice have not been reported up to now.

6 Regulatory Mechanisms: Receptorial, Transcriptional, and Transductional As described, VGCCs are comprised of five different protein subunits: a1, a2d, b, and g [118]. The a1 and a2 subunits were co-purified with equimolar stoichiometry [106, 118]. The a2d subunit was first discovered in rabbit skeletal muscle

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[27] and later in the rat brain [67] and human brain [129]. The a2d subunits are derived from a single gene [27], and alternative splicing mechanisms have been proposed to give rise to variants of the a2 subunit [67]. Other details of the protein subunits comprising VGCCs are summarized in Table 1. There are several means by which the levels of calcium channels are regulated by second messenger pathways, such that L-type calcium channels are upregulated by cAMP-dependent kinase [98]. The regulation of N- and P/Q-type channels are inhibited by the activation of heterotrimeric G-proteins by Gprotein-coupled receptors (GPCRs). GPCRs involved in this regulation include two adenoreceptors, m- and d-opioid receptors, GABA-B receptors, and adenosine A1 receptors. These receptors or GTP analogues slow the current activation kinetics of N- and P/Q-type channels.

7 Physiological Properties VGCCs play a major role in both normal functioning and various pathological processes. The calcium action potential and subsequent calcium currents are detected in mammalian skeletal, cardiac, and all excitable cells. For example, depolarization of smooth muscle opens L-type calcium channels and causes an influx of extracellular Ca2þ, which binds calmodulin. The activated calmodulin molecule activates myosin light-chain kinase, which phosphorylates myosin. Phosphorylated myosin form cross-bridges with actin thin filaments, and thus contraction of smooth muscle fibers is driven by the sliding filament mechanism. L-type VGCCs on the neuronal cell body provide the Ca2þ signals necessary for gene activation, cell survival, and protein expression. Calcium channels are also present at low levels in many non-excitable cells, such as immune cells, though their role has not been elucidated [12].

8 Pathophysiological Relevance in Stroke The level of intracellular Ca2þ concentration is strictly regulated by multiple complex machineries. Intracellular Ca2þ normally binds to calcium-binding proteins, such as calmodulin, or exists in storage in cellular organelles, such as the endoplasmic reticulum (ER) and mitochondria. Free Ca2þ released into the intracellular space is actively excreted by the Naþ/Ca2þ-exchange system, which is coupled to ATPase. However, cerebral ischemia causes breakdown of the regulatory mechanisms, and unrestricted increases in the concentration of calcium ions in the intracellular space cause cell death. Excessive influx of Ca2þ via glutamate receptors, and excessive release of Ca2þ from the ER are involved in various cytotoxic processes, such as proteolysis, oxidative damage, and apoptosis. Although the main inflow of calcium into neurons in ischemia is mediated by


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glutamate receptors, VGCCs also participate in Ca2þ entry. Thus, the L-type VGCC activator Bay K 8644 enhances and the blocker nimodipine inhibits cellular apoptosis and mitochondrial damage, suggesting that Ca2þ entry through L-type channels is responsible for apoptosis [13, 55]. L-type VGCCs also ensure an early rise in intracellular calcium in ischemia. L-type Ca2þ channels are upregulated in reactive astrocytes after ischemia [127]. Upregulation of L-type Ca2þ channels in reactive astrocytes may contribute to the maintenance of ionic homeostasis in injured brain regions by increasing the release of neurotrophic factors to promote neuronal survival and differentiation, and enhancing signaling in astrocytic networks in response to injury [127]. Inhibiting L-type VGCCs with dihydropyridine abolished the rise in intracellular calcium [94]. L-type Ca2þ channels are also involved in the activation of calcium/calmodulin-dependent protein kinase II (CaMKII), which induces serine phosphorylation of GluR6 and neuronal cell death, and nifedipine can exert neuroprotective effects in this way [43]. Transient increases in N-type Ca2þ channels are typically observed 2–3 days after ischemia [22]. Delayed neuronal death in the CA1 region after 5 min of ischemia is attenuated by o-conotoxin in a dose-dependent manner [130]. oConotoxin has a neuroprotective effect against ischemic injury, probably by inhibition of N-type VGCC [22]. Several synthetic o-conopeptides, such as synthetic MVIIA (SNX-111), have allowed the evaluation of the therapeutic potential of blocking N-type Ca2þ channels in a variety of pathological conditions, including cerebral ischemia [78, 103]. T-type calcium channel inhibitors also reduce ischemic cell damage [87]. Although the inhibition of L-type VGCCs with dihydropyridine abolished the rise in intracellular calcium usually observed after 2–4 min, it had no effect on a small very early increase in intracellular calcium concentration [94]. Thus, the potential role of low-voltage-gated T-type calcium channels during brain ischemia has been suggested. Because T-type calcium channels have abilities to sustain a continuous calcium influx in neurons and glia at rest states by a window current mechanism, the increase in early intracellular calcium concentration observed after the initiation of an ischemic insult could be the expression of an unbalanced equilibrium between sustained calcium influx through T-type calcium channels and dysfunctional efflux mechanisms under metabolic distress [87]. Therefore, pharmacological inhibition of the T-type calcium current by mibefradil, kurtoxin, nickel, zinc, and pimozide during the oxygen–glucose deprivation episode could provide significant protections against delayed neuronal death [87].

9 Pharmacological Modulation The pharmacology of drugs acting on calcium channels is quite distinct. The calcium channel blockers used widely in the therapy of cardiovascular diseases are derivatives of dihydropyridines that target the Cav1 (L-type) channels, and

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act at three separate, but allosterically coupled, receptor sites [37]. Dihydropyridine calcium channel blockers are often used to reduce systemic vascular resistance and arterial pressure. These include ‘‘-dipine’’ drugs, such as amlodipine, felodipine, nifedipine, nicardipine, and nimodipine. Phenylalkylamine calcium channel blockers, such as verapamil, are relatively selective for myocardium, reduce myocardial oxygen demand and reverse coronary vasospasm, and they are often used to treat angina. Phenylalkylamines are intracellular pore blockers that are thought to enter the pore from the cytoplasmic side of the channel and block it [50, 52, 113]. The receptor site of dihydropyridine is closely apposed to that of phenylalkylamine, even though they share some common amino acid residues. Diltiazem and related benzothiazepines are thought to bind to a third receptor site, even though the amino acid residues required for their binding largely overlap with those required for phenylalkylamine binding. The CaV2 subfamily (P/Q, N, and R-type VGCC) of calcium channels is relatively insensitive to dihydropyridine calcium channel blockers. Several newly developed anti-epileptic drugs such as lamotrigine, levetiracetam, and pregabalin have some affinities to these receptors, which will be discussed below. The CaV3 subfamily (T-type) of calcium channels is insensitive to dihydropyridines and toxins, and there are no widely useful pharmacological agents that block T-type calcium currents [92]. Mibefradil is somewhat selective for T-type over L-type calcium currents (threefold to fivefold). Development of more specific and high-affinity blockers of the CaV3 family would be useful for both therapy and research.

10 Preliminary Clinical Trials Many clinical trials with Ca2þ antagonists acting on VGCCs have been conducted for neuroprotection. However, a critical and extensive review has shown that Ca2þ antagonists do not reduce the risk of death or dependency after acute ischemic stroke [54]. The L-type Ca2þ channels blocker nimodipine is one such example. Initially, a randomized, placebo-controlled trial in stroke patients showed a significant reduction in death and neurological impairment after administration of the calcium antagonist nimodipine [36]. Similar results were found in subarachnoid hemorrhage, in which administration of nimodipine before the onset of ischemia was associated with a reduced occurrence of cerebral ischemia and improved clinical outcome [31]. However, further randomized studies with nimodipine or other calcium antagonists (e.g., flunarizine, isradipine) in ischemic stroke did not confirm the beneficial effect of the earlier study [124]. However, subgroup analyses by the American Nimodipine Study Group of patients treated with nimodipine within 18 h after stroke onset in the American Nimodipine Trial suggested an improved outcome after treatment [1]. The


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notion that a short time interval between stroke onset and the start of treatment is crucial was supported by results of a meta-analysis of nine trials of nimodipine in patients with ischemic stroke [82]. Although the overall analysis did not show any beneficial effect, there was a statistically significant reduction in poor outcome in patients treated with nimodipine within 12 h after stroke onset (odds ratio, 0.62; 95% CI, 0.44–0.87) [82]. Though this raised hope that the risk of death and neurological impairment after ischemic stroke could be reduced with proper treatment, a systematic review of the effects of calcium antagonists in stroke could not confirm this positive effect of early treatment [54]. Another study, the Very Early Nimodipine Use in Stroke (VENUS) trial was designed to test the hypothesis that early treatment with nimodipine has a positive effect on survival and functional outcome after stroke [53]. In this randomized, double-blind, placebo-controlled trial, treatment was started by general practitioners or neurologists within 6 h after stroke onset (oral nimodipine 30 mg QID or identical placebo, for 10 days). However, the results of the VENUS study did not support the beneficial effect of early nimodipine treatment in stroke patients [53]. Though there have been other clinical trials involving the use of other Ca2þ channel blockers, such as isradipine [5], flunarizine [34, 72], and nicardipine [73], the overall systemic review and the individual results rule out a clinically important effect of administration of a calcium channel antagonist after ischemic stroke [55]. Therefore, the use of L-type VGCC blockers in acute stroke was not accepted. Because all of these effort-consuming trials were based on insufficient preclinical designs and evidences, these results raised a lot of discussion concerning the focus of future neuroprotective preclinical experiments. However, most of the clinical trials have used L-type Ca2þ channel antagonists, such as nimodipine. On the other hand, the clinical benefits of antagonists that selectively modulate N- or P/Q-type channels have not been systemically explored.

11 Therapeutic Perspectives As discussed above, the clinical benefits of antagonists that selectively modulate Nand P/Q-type VGCCs should be investigated in future studies. Interestingly, various anti-epileptic drugs have potent inhibitory effects on N- or P/Q-type channels. Lamotrigine, which has been known to inhibit Naþ-dependent repetitive firing discharge, exerts a potent inhibitory effect on N-type VGCCs [126]. Neuroprotective effects of lamotrigine have been reported in a rat cerebral ischemia model [108, 128], although some controversy remains concerning the efficacy of lamotrigine [122]. The severity of the ischemic insult as well as the differential time schedule should be taken into account when conducting a clinical trial. Another feasible anti-epileptic drug in neuroprotection is levetiracetam, which modulates N-type Ca2þ channels [76]. The neuroprotective effect of levetiracetam is comparable to that of the NMDA antagonist MK-801, with less adverse effects

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[42]. Gabapentin also modulates P/Q-type and L-type channels in addition to its inhibitory action on Naþ-dependent firing [111]; however, its neuroprotective effect in cerebral ischemia has not been elucidated. The drugs gabapentin and pregabalin interact with the P/Q-type VGCCs and have alleviated ataxia in cerebellar atrophy and other degenerative ataxias [35]. Pregabalin has exhibited more delayed channel inactivation and greater antiepileptic power than gabapentin under experimental conditions [7, 38]. Applications of gabapentin and pregabalin on VGCCs together with a possible anti-glutamatergic action [7] suggest that they may contribute to neuroprotection against ischemia. Thus, further efforts to develop therapeutics for cerebral ischemia targeting Nor P/Q-type VGCCs are still warranted. In addition, studies should develop combination therapies of VGCC blockers with other neuroprotective agents that can be used in the early treatment of ischemic stroke, such as nitrite [62], and that orchestrate other Ca2þ-permeable ion pores involved in cerebral ischemia, such as glutamate receptors, glutamate transporters [21], and Naþ/Ca2þ exchangers [58].

12 Conclusions In this review, we discussed the characteristics of VGCCs and the therapeutic feasibility of VGCC antagonists in ischemic stroke. Although L-type Ca2þ channel blockers (i.e., nimodipine) have failed to attenuate neurologic deterioration in large clinical trials, a number of newly developed Ca2þ channel blockers that target non-L-type channels remain uninvestigated. Because ‘‘calcium’’ is the key regulator in ischemic injury and neuronal death, continuing efforts to discover clinically effective therapeutics based on VGCCs and/or calcium modulators are strongly warranted. Scientists and clinicians around the world are waiting for such data to be reported soon.

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The Diverse Roles of Kþ Channels in Brain Ischemia Hiroaki Misonou and James S. Trimmer

1 Introduction Brain cells express a wide variety of Kþ-selective ion channels. Among these are inward rectifying Kþ (Kir) channels, Ca2þ-activated Kþ channels, and voltagegated Kþ (Kv) channels. These Kþ channels are crucial regulators of membrane excitability due to their ability to stabilize the membrane potential. During ischemic stress in the brain a variety of pathological events occur, including increased extracellular levels of glutamate, increased levels of intracellular Ca2þ and Naþ, and a rapid reduction in intracellular ATP levels [1]. These events and the associated aberrant neuronal membrane excitability ultimately lead to neuronal damage and death. We have learned in the previous chapters that numerous molecular events are implicated in the deleterious effects of brain ischemia. However, brain neurons also contain numerous endogenous protective mechanisms [1]. Kþ channels are ideally suited for this job. KATP/Kir6.2 channels open in response to reduced cellular ATP levels or [ATP]i, Slo1 channels exhibit enhanced activation in response to increased intracellular Ca2þ levels or [Ca2þ]i, and the activity of Kv2.1 channels can be potentiated by either of these changes. The opening of these Kþ channels hyperpolarizes the membrane potential toward the Kþ equilibrium potential (EK) and leads to a suppression of neuronal excitability. Recent studies have revealed the importance of protective mechanisms provided by endogenous Kþ channels in the response of neurons to brain ischemia.

2 Gene Structures of Kir6.2, Slo1, and Kv2.1 The human Kir6.2 gene (KCNJ11) is located on chromosome 11 (11p15.1) and is quite simple in that the entire coding region (1,173 bp) is contained within a single exon. The human Slo1 gene (KCNMA1) is located on chromosome 10 H. Misonou (*) Department of Neural and Pain Sciences, Program in Neuroscience, Dental School, University of Maryland, Baltimore, MD 21201, USA e-mail: [email protected]


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(10q22.3) and is quite complex in that the coding sequence is spread across 770 Kbp in multiple (>20) exons. Slo1 transcripts are subjected to extensive processing, and mammalian neurons express alternatively spliced variants [2; 3]. The human Kv2.1 gene (KCNB1) is located on chromosome 20 (20q13.11), and the 2,577 bp coding region is contained in two exons within the 109 Kbp gene.

3 Protein Structures of Kir6.2, Slo1, and Kv2.1 Kþ channels are tetramers of pore-forming a subunits. The 390 amino acid Kir6.2 a subunit polypeptide contains two transmembrane segments and cytoplasmic N- and C-termini. Although a tetramer of Kir6.2 subunits confers the core of the channel and the primary ATP-binding site, the sulfonylurea receptor SUR1 subunit is a component of all brain Kir6.2-containing channels, which exist as [Kir6.2]4[SUR1]4 octameric KATP channel complexes [4]. The-858amino acid Kv2.1 a subunit contains a cytoplasmic N-terminus, six transmembrane segments, and a cytoplasmic C-terminus. The tetrameric assembly of the transmembrane segments including the pore-forming loop creates the core of the Kv channel [5]. The basic configuration of Slo1 channels (1,200 residues) is similar to that of Kv2.1 except that Slo1 has an additional transmembrane segment (S0) before the core region such that the N-terminus of Slo1 is exposed to the extracellular space [6]. Slo1 has an extremely long cytoplasmic C-terminus that contains components of the Ca2þ-sensing mechanism [7].

4 Molecular Biology of Kir6.2, Slo1, and Kv2.1 Kir6.2 was cloned from a human genomic library [8], and shares 96% amino acid identity with mouse and rat Kir6.2. Kir6.2 is mutated in patients with hyperinsulinemic hypoglycemia and permanent neonatal diabetes. Slo1 was originally isolated from a mouse brain cDNA library screened with a Drosophila Slowpoke cDNA probe [9]. Human, mouse, and rat Slo1 polypeptides are highly similar. A point mutation in human Slo1 leads to generalized epilepsy and paroxysmal dyskinesia. Kv2.1 was originally cloned from a rat brain cDNA library by expression cloning in Xenopus oocytes [10]. Mouse (97% amino acid identity) and human (94% amino acid identity) are highly similar to rat Kv2.1. Mutations in Kv2.1 have not been associated with any diseases.

5 Tissue and Cellular Distribution of Kir6.2, Slo1, and Kv2.1 in Mammalian Brain Kir6.2, Slo1, and Kv2.1 are highly expressed in mammalian brain, although they are also expressed in other tissues. Most studies of their localization have been performed in rat or mouse brain. Kir6.2 is expressed widely in brain, as is

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SUR1 [11; 12]. Immunohistochemical studies suggest that Kir6.2 is mainly found in the somata and dendrites of brain neurons [13; 14]. Slo1 is also widely expressed in the brain, and is predominantly found in neuronal axons and nerve terminals [15; 16]; this is especially apparent in the hippocampus (Fig. 1) and the cerebellum (Fig. 2). Slo1 is also found in dendrites, for example in cerebellar Purkinje cells (Fig. 2) [16]. Kv2.1 is highly expressed in virtually all neurons in the central nervous system (Fig. 1) [17]. Kv2.1 is highly concentrated in the somata and the proximal portion of dendrites (Fig. 1) where it forms discrete surface clusters [18].

Fig. 1 Localization of Slo1 and Kv2.1 in adult rat hippocampus. Double immunofluorescence staining for Slo1 (red) and Kv2.1 (green). DG: dentate gyrus; PF: perforant path; MF: mossy fibers

Fig. 2 Localization of Slo1 in the axons and terminals of cerebellar basket cells and the dendrites of Purkinje cells. Rat cerebellar sections were stained by double immunofluorescence for Slo1 (red) and phosphorylated forms of neurofilament H (as an axonal marker, green). Slo1 is highly detected not only in the basket cell terminals attached to the cell bodies of Purkinje cells, but also in Purkinje cell dendrites in the molecular layer of the cerebellar cortex

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Kir6.2 [19] and Slo1 [20] channels are also present in mitochondria where they may play a distinct role in hypoxic stress. This aspect of their function will not be discussed here.

6 Biophysical Properties of Kir6.2, Slo1, and Kv2.1 Channels Most biophysical studies on Kþ channels have been performed in heterologous expression system such as Xenopus oocytes or transfected mammalian cell lines. KATP channels formed by heterologously expressed Kir6.2 and SUR1 have a relatively large Kþ conductance of 70 pS [21]. In the presence of [ATP]i > 500 mM, the open probability of Kir6.2/SUR1 channels is nearly zero but increases as [ATP]i decreases [4]. Although Kir6.2 is a member of the Kir channel family whose ionic currents are blocked at positive membrane potentials, Kir6.2 channels display only weak rectification and are able to exhibit substantial outward Kþ currents above EK [22]. This property would allow Kir6.2 channels to confer ischemia-induced outward Kþ currents in neurons. Slo1 channels are exceptional in their large unitary conductance, which ranges from 100 to 270 pS [7]. When activated by membrane depolarization coinciding with increased [Ca2þ]i, Slo1 channels show non-inactivating Kþ currents similar to the delayed rectifier current. Both voltage and Ca2þ dramatically regulate the open probability of Slo1. In the absence of Ca2þ, Slo1 is not active within the physiological range of membrane potentials. The open probability of Slo1 channels dramatically increases when [Ca2þ]i is 1–100 mM, yielding activation at physiological membrane potentials. Kv2.1 forms delayed rectifier Kþ channels with a single channel conductance of 10pS [23] and a relatively high-threshold for voltagedependent activation (G1/2 0 to +20 mV), although this is highly dependent on Kv2.1 phosphorylation state [39]. The steady-state inactivation of Kv2.1 is unique that the voltage-dependent curve is U-shaped, with maximum inactivation at 0 mV and less inactivation observed with larger depolarizations [24]. It should be noted that these characteristics are similar to those of somatic delayed rectifier currents in hippocampal pyramidal neurons [25], of which Kv2.1 is thought to be the major component [26; 27].

7 Regulation of Kir6.2, Slo1, and Kv2.1 Channels The activity of KATP channels is highly regulated by the metabolic state of the neuron, as the channel is inhibited by the binding of ATP and activated by the binding of Mg2þ-nucleotides such as Mg2þ-ADP [4]. The binding of ATP to Kir6.2 stabilizes the closed state of the channel [28]. Because the association


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constant for ATP binding to KATP channels is 10 mM, and given a normal [ATP]i 1 mM, most of the Kir6.2 subunits are normally bound to ATP. Thus, under physiological conditions the open probability of KATP channels is negligible. Reducing [ATP]i results in loss of bound ATP from Kir6.2 and association of Mg2þ-ADP with SUR1, leads to activation of Kir6.2-containing KATP channels. Kir6.2 also binds and is regulated by the important membrane phospholipid PIP2, which is mutually exclusive with the ATP binding and stabilizes Kir6.2 in the open conformation [28]. Kir6.2 is also modulated by protein kinase A [29]. Slo1 channels can be activated by either membrane depolarization or increased [Ca2þ]i alone. Under physiological conditions, Slo1 channels need to be activated synergistically by both membrane depolarization and Ca2þ, and can act as coincidence detectors of these two important neuronal signaling events. However, in pathological conditions where [Ca2þ]i can be extremely high, Slo1 may be activated solely by Ca2þ. Slo1 is also subjected to phosphorylation by multiple kinases [30]. Recent mass spectrometric analyses of Slo1 purified from mammalian brain have revealed extensive phosphorylation on its cytoplasmic C-terminus [3]. Native Slo1 channels are associated with the auxiliary b subunits, b1–4 [31]. Intriguingly, not only do the b subunits alter the biophysical properties of Slo1 channels, but they also confer sensitivity to various steroid hormones [32], including estradiol [33], which has been implicated as a protective agent in brain ischemia. Slo1 channels are sensitive to redox state, such that a reduction in oxygen in chemoreceptors in the carotid body and in heterologous expression systems leads to inhibition of Slo1 [34]. In contrast, oxidation of Slo1 polypeptides, which could occur during reperfusion following ischemia, can activate Slo1 by shifting its voltage dependence of activation by –30 mV [35]. The contribution of the redox regulation of Slo1 in ischemia is unclear as these effects are negligible in the presence of Ca2þ above 100 mM, which is readily achieved in ischemic neurons. Heme can bind directly to Slo1 [36], conferring regulation of Slo1 by carbon monoxide [37]. Kv2.1 is activated solely by voltage. However, its voltage-dependent properties are dramatically altered by changes in Kv2.1 phosphorylation state [38]. Recent mass spectrometric analyses have revealed that Kv2.1 is constitutively phosphorylated at multiple sites (>16) in brain neurons [39]. Individual mutations of the phosphorylation sites to dephosphorylation-mimetic alanine result in significant shifts in the voltage dependence of activation and steady-state inactivation toward more hyperpolarizing membrane potentials. Interestingly, concurrent mutations of multiple sites to alanine elicit an additive effect, indicating that Kv2.1 channels are subjected to a graded regulation by changes in phosphorylation at multiple sites. Similar graded responses have also been observed for the delayed rectifier current in neurons [38], where the Ca2þ/ calmodulin-dependent protein phosphatase calcineurin plays a crucial role by dephosphorylating Kv2.1 [39; 40].

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8 Physiological Properties of the KþChannels Studies of Kir6.2 knockout mice revealed that, under physiological conditions, the firing behavior of glucose-responsive neurons in the hypothalamus is regulated by the action of Kir6.2 [41]. These hypothalamic neurons increase their firing rate when the extracellular glucose level is elevated (from a few to tens of millimolar). This behavior was completely eliminated in the Kir6.2 knockout mice, indicating that the inactivation of Kir6.2 accounts for the change in the firing phenotype. However, the exact mechanism is unknown as intracellular ATP can efficiently block Kir6.2 with an IC50 10 mM. It seems unlikely that the [ATP]i in these neurons fluctuates between the micromolar and millimolar range, such that other regulatory mechanisms, such as PIP2 binding, may be involved. The sensitivity of Slo1 to Ca2þ, the channel’s rapid activation kinetics, and its extremely large single-channel conductance makes activation of Slo1 an important response to Ca2þ entry and membrane depolarization under physiological conditions. Slo1 is implicated in the generation of the fast phase of the after-hyperpolarization potential in the somata of pyramidal neurons [42], and acts as a key determinant of the refractory period of action potential firing [43]. At dendrites and nerve terminals, Slo1 channels can limit the influx of extracellular Ca2þ through voltage-gated Ca2þ channels [44; 45]. These characteristics might also be important in the pathological state of neurons during brain ischemia. Kv2.1 channels have relatively slow activation kinetics [46] and thus may not be efficiently recruited during a single action potential in the soma. However, in response to repetitive firing of action potentials and frequency-dependent action potential broadening observed in many neurons [43], Kv2.1 can be effectively activated by Ca2þ-dependent dephosphorylation and contribute to the repolarization of membrane potential. In fact, antisense knockdown of Kv2.1 in hippocampal pyramidal neurons results in aberrant hyperexcitability of the neurons and resultant Ca2þ overload at high firing frequencies [26]. As the history of activity within neurons can affect the phosphorylation state of Kv2.1 (higher activity leading to dephosphorylation and enhanced activation of Kv2.1, lower activity the opposite) [38], the role of Kv2.1 in cell excitability is conditional.

9 Pathophysiological Relevance in Stroke For most neurons under physiological conditions, Kir6.2/SUR1 channels are presumably closed due to high [ATP]i. However, they can be rapidly activated in various in vitro models of brain ischemia. Mimicking ischemia in vitro rapidly ( 1 mM, suggesting that this compound could selectively target Slo1 channels in cells having elevated [Ca2þ]i. Based on the assumption that BMS-204353 could provide specific activation of Slo1 channels in ischemic neurons, BMS-204353 had been tested in the preclinical studies using rat models of brain ischemia. BMS-204353 showed a promising protective effect against the focal ischemia in


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reducing the infarct volume at 24 h after onset. Although Slo1 is highly expressed in vascular smooth muscle cells, the drug showed a minimal effect on vascular smooth muscle. BMS-204353 has more recently been tested in a phase III clinical trial (see below). Kv2.1 is also sensitive to TEA, but at millimolar concentrations, and is relatively insensitive to 4-aminopyridine (4-AP). There are no specific small molecule Kv2.1 modulators, although a number of spider toxins, including hanatoxin and stromatoxin, have been shown to act as gating modifiers of Kv2.1, as well as Kv2.2 and Kv4 channels [65].

11 Preliminary Clinical Trials KATP channel modulators such as glibenclamide have been used for years for the clinical treatment of diabetes. A recent study suggests that prior use plus post-event use of sulfonylurea agents could have a beneficial effect on stroke outcome [66]. After the discovery of the promising protective effect of the Slo1 channel opener, BMS-204352, in animal models of brain ischemia [64], the drug was subjected to a phase III clinical trial [67]. Unfortunately, there was no significant effect of the drug over placebo. No clinical trials for Kv2.1 have been undertaken.

12 Therapeutic Perspectives The rationale of targeting Kir6.2, Slo1, and Kv2.1 would be to moderate the pathological hyperexcitability of ischemic neurons and thereby increasing the survival rate of the affected neurons after an ischemic event. The major challenge in targeting these Kþ channels in therapeutic treatments is to develop compounds with sufficient specificity to localize the effect of drugs to ischemic neurons, because all of these Kþ channels are expressed widely in the brain and throughout the body. Although the clinical trial ultimately failed [67], the Slo1 opener BMS-204352 provides valuable insights into what may constitute a model drug for brain ischemia. Biophysical analyses of the drug action revealed that enhanced Slo1 channel activation occurs only at high [Ca2þ]i [64]. Therefore, this compound would be expected to elicit maximal effects in ischemic neurons where [Ca2þ]i is high relative to unaffected neurons and non-neuronal cells. Coupling the efficacy of a drug with the changes that occur in response to ischemia, such as a rise in [Ca2þ]i and a reduction in ATP, may allow for future development of drugs with specificity for ischemic neurons. One caveat of using Kþ channels as drug targets is that excessive activation of Kþ channels may have deleterious effects on neurons. Prolonged (1 h) ischemic insults, as well as other neurodegenerative insults, result in increased levels of delayed rectifier currents in cultured neurons [68], likely via enhanced

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activity of Kv2.1 [69]. Intriguingly, TEA blocks the delayed neuronal death after ischemic insult [70], indicating that the excess efflux of Kþ may trigger a signaling mechanism leading to neuronal death. Although the mechanism underlying the deleterious effect of Kþ channel activation is not fully understood, therapeutic approaches resulting in sustained enhancement of neuronal Kþ channels should be carefully considered. The aforementioned strategy of linking a drug’s efficacy to intracellular changes associated with ischemia should be effective not only for localizing the effect but also for limiting its duration. Detailed biophysical studies of the mechanism of action of channel modulators remain crucial to the development of effective new therapeutic strategies targeting brain ischemia. Acknowledgments Work from our laboratories cited above was supported by NIH (NS34383 and NS42225) and AHA.

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Clinical Trials with Drugs Targeting Ionic Channels, Antiporters, and Pumps in Ischemic Stroke Alessandra Spinali, Giuseppe Pignataro, Gianfranco Di Renzo, and Lucio Annunziato

Despite the great concerted efforts to develop effective therapies for brain ischemia, the only successfully available therapy for acute stroke treatment is the recombinant tissue plasminogen activator (rt-PA). During the last decades, several reproducible and controlled animal models of brain ischemia have been generated and validated in an attempt to develop new effective therapies for stroke treatment. Experimental studies using neuroprotective strategies in these models have provided (1) incontrovertible proof of principle that high-grade protection of ischemic brain is an achievable goal and (2) preclinical evidence supporting potential neuroprotective efficacy [19]. However, though the preclinical evaluation of several neuroprotective agents has fostered high expectation of their clinical efficacy, translation of neuroprotective benefits from the laboratory to the clinical setting has not been successful [6]. Several preclinical and clinical factors can be considered responsible for the unsuccessful outcomes of the past clinical trials in stroke.

1 Factors to Be Considered in Preclinical Studies The main cause that has probably rendered unsuccessful the clinical trials for stroke so far has been the lack of sufficiently solid evidence-based preclinical findings. In fact, there are at least three minimal preclinical requirements to be fulfilled before an agent can be fully approved for use in human trials. First, it is necessary to demonstrate that the activity of the compound is provided with robust and clinically relevant perspectives of efficacy. Second, a proper experimental study design should be planned. Third, the confirmation of positive findings by other laboratories must be provided [6, 19, 40]. However, most of the clinical trials with neuroprotectant agents have been L. Annunziato (*) Division of Pharmacology, Department of Neuroscience, ‘‘Federico II’’ University of Naples, Via Pansini 5, 80131 Naples, Italy e-mail: [email protected]


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often launched without fully satisfying these preclinical requirements [19]. In addition to these shortcomings, a great number of variables could have negatively influenced the quality, consistency, and successful outcome of the preclinical studies. Among the most relevant of these variables are [19] the following: (a) animal-related factors: age (young but not old), sex (male but not female), comorbidities (healthy but not ill), species, and strain; (b) anesthetic agent used and co-administered drugs; (c) extent and consistency of physiological control of vital parameters; (d) model-related factors: focal versus global ischemia, transient versus permanent occlusion, method of vascular occlusion, duration and intensity of ischemia, duration and extent of reperfusion, survival time after the insult, and behavioral and pathological sequelae; (e) outcome assessment; (f) quality of study design: randomization, blinding, and analysis of the data; (g) specific properties of neuroprotective agents: stability, dose, route of administration, pharmacokinetics and pharmacodynamics, and time of administration with respect to ischemic insult (i.e., before or just immediately after the ischemic insult, in contrast to the therapeutic window of the human pathology).

2 Factors to Be Considered in Clinical Studies The main reason why clinical trials often fail to replicate the same safety and efficacy profiles of drugs tested in animal models is that the reality of clinical settings cannot reproduce the same laboratory scenarios. This is particularly true in terms of time of treatment, outcome measures, functional assessments, drug dose-regimens, and, finally, comorbidity conditions. We will now briefly describe each of these factors separately in an effort to better elucidate the reasons of their unsuccessful results in clinical trials in stroke patients. (a) Time of treatment. One of the most inconsistent factors between preclinical and clinical studies has been the time when treatment begins. Indeed, whereas in many animal studies neuroprotective agents have been given before or immediately after the onset of ischemia, in most clinical trials the time window has been beyond 3 h [6, 19]. (b) Outcome measures. The approaches to detecting the treatment outcomes have also differed considerably. Indeed, in most preclinical studies, the efficacy of neuroprotective agents has been detected by measuring the reduction of histological infarction volume. By contrast, in clinical trials neuroprotective efficacy is measured by assessing the patient’s neurological function using the NIH Stroke Scale or the modified Rankin Scale. In fact,

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infarct volumes poorly correlate with the functional outcome. Moreover, most animal stroke models used in preclinical neuroprotection studies are MCA occlusion models, whereas patients enrolled in clinical trials are often affected by damage to different brain regions. Thus, some animal models have proven to be poor predictors of clinical trial results [6, 19]. (c) Functional assessment. The reason why functional assessment may have affected the therapeutic outcomes in the clinical setting, as opposed to lab trials, is that in animal studies with neuroprotective agents, the neurocognitive function has been evaluated through different tests chosen by researchers according to their own experience. In clinical trials, although various measures have been used to determine mental and physical impairment or disability, none of these clinical scales has proven to correlate with infarct volumes or with any particular battery of preclinical functional tests [6, 19]. (d) Drug dose-regimens. To avoid toxicity, in some clinical trials a dose significantly lower than that shown to be efficient in animal studies has been administered. In addition, administration schedules for treating patients has not exactly reproduced treatment regimens used in animal studies [6, 19]. (e) Comorbidity conditions. Finally, comorbidity conditions of patients can have deeply limited the success of the therapy. In stroke models, researchers usually choose healthy young male animals. By contrast, stroke patients enrolled in ischemia clinical trials are usually old and suffer from multiple chronic diseases such as atherosclerosis, hypertension, diabetes, hyperlipemia, and prior stroke. The combination of these conditions could have eventually affected functional outcomes, thus altering the measurements of drug efficacy and safety of neuroprotectants [6, 19]. In addition, what may also have contributed to the failure of the clinical efficacy of neuoprotectants is the fact that in most cases these drugs have been tested on a number of patients by far insufficient to demonstrate clinical efficacy and without a rigorously sophisticated clinical-trial methodology [19]. Therefore, in light of these considerations, we suggest that future clinical trials involving neuroprotective agents should be designed by taking into consideration evidence derived from preclinical studies along with current guidelines for drug experimentation in stroke. These should include the following [6, 19]:

A time window of treatment compatible with the preclinical findings should be attempted, to maximize possible therapeutic benefits. If the drug is proven to be beneficial, follow-up trials with an extended therapeutic window may be designed. Drug regimens chosen for phase III efficacy studies should be compatible with preclinical doses and selected on the basis of phase I and II clinical studies. Patient selection should reflect the therapeutic target. In fact, the mechanism of injury elicited by ischemic stroke may vary because of the different brain region vulnerability. CT scans and various MRIs may be useful tools, but this has not yet been proven. Moreover, it is important to consider the severity of the stroke that may affect the study outcome. Adequate follow-up periods should be scheduled.

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Choice of clinically meaningful primary-outcome measures should be established.

Sample size should be powered to demonstrate efficacy. Choice of stratification variables should be relevant to the agent in question. Combination therapy should be taken into account. Thrombolytic therapy with rt-PA has become the standard of care. Various families of neuroprotective agents, used in combination with rt -PA, may have synergistic effects against ischemic injury. For this reason, the dose of each drug may be reduced to limit toxicity and increase tolerability; however, combining two or more drugs in the same study would render the trial design more complex. In the following tables (1–9) we will consider, among the wide array of drugs tested in humans for efficacy in stroke intervention, all clinical trials related to agents targeting ionic channels, such as calcium channel blockers, GABA antagonists, glutamate receptor antagonists, sodium channel blockers, and potassium channel openers [25]. Table 1 Clinical trials with drugs targeting ionic channels in ischemic stroke Drug class Number of clinical trials Calcium channel blockers Nimodipine Flunarizine GABA agonists Clomethiazole Glutamate receptor antagonists AMPA antagonists o YM 872 o ZK-200775 NMDA antagonists o Competitive NMDA antagonists & Selfotel o NMDA channel blockers & Aptiganel & Dextrorphan & Magnesium & NPS-1506 & Remacemide o Glycine site antagonists & ACEA 1021 & GV 150 526 Sodium channel blockers Fosphenytoin Lubeluzole Sipatrigine Potassium channel openers BMS-204352

13 2 2

2 1

2 2 1 4 1 1 1 5 1 5 3 1

Within 48 h

Within 24 h

Phase III, randomized, double-blind, placebocontrolled, multicenter clinical trial of 1,064 patients.

Prospective, multicenter, double-blind, randomized, placebocontrolled trial of 186 patients.

ANS. American Nimodipine Study [45]

Controlled trial of nimodipine in acute ischemic stroke [17]

Nimodipine

Name of the study

4 weeks

21 days

30 mg every 6 h.

60 mg, 120 mg, and 240 mg daily orally.

Table 2 Clinical trials with drugs blocking calcium channels Status of the trial: clinical Treatment: phase and number of time from Treatment patients onset duration Doses Nimodipine had no overall effect when treatment was begun within 48 h. Post hoc analysis suggested benefits for the subgroup treated with 120 mg nimodipine within 18 h, and who had negative computed tomographic scans. Mortality from all causes in the 4-week treatment group was significantly reduced with nimodipine as compared with placebo but the improvement in survival was restricted to men. A significantly better neurologic outcome was also observed in the nimodipine group. The improvement in neurological status was greatest in patients with a moderate to severe deficit at baseline.

Comments

Clinical Trials with Drugs Targeting Ionic Channels 229

Effect of nimodipine on regional cerebral glucose metabolism in patients with acute ischemic stroke as measured by positron emission tomography [23]

Double-blind study of nimodipine in non-severe stroke [4]

Name of the study

Phase II, single-center, double-blind, randomized, placebocontrolled pilot study of 60 patients. Randomized, doubleblind, placebocontrolled study of 27 patients.

Status of the trial: clinical phase and number of patients 14 days

21 days

Within 48 h

Within 48 h

Table 2 (continued) Treatment: time from Treatment onset duration

2 mg/h continuous infusion followed by 120 mg/day orally.

30 mg q.i.d. orally.

Doses

Treatment with nimodipine was well tolerated but Mathew scale scores did not differ between the two groups. The Barthel Index at the end of treatment was lower in the nimodipine group but the difference was not significant. The nimodipine group, however, showed the larger trend toward subsequent improvement. No significant differences were found in Mathew Scores. The two groups were similar in their initial metabolic depression as measured by FDG PET but glucose metabolism in non-infarcted regions increased more in the nimodipine than in the control group.

Comments

230 A. Spinali et al.

Within 48 h

Within 24 h

Randomized, doubleblind, placebocontrolled trial of 164 patients.

Randomized, multicenter, double-blind, placebocontrolled trial of 295 patients.

INWEST. Intravenous Nimodipine West European Stroke Trial [48]

21 days

6 months


Intravenous nimodipine in acute ischemic stroke [39]

Name of the study

Status of the trial: clinical phase and number of patients

1 or 2 mg/h intravenous infusion for 5 days followed by 120 mg/day orally for 16 days.

2 mg/h intravenous infusion for 10 days, followed by 180 mg/day orally for 6 months

Doses

Comments There were no differences between treatment groups in regard to deaths, Toronto stroke scale scores, or functional disability. There was, however, a trend toward effectiveness of nimodipine in patients treated within 12 h of stroke onset. The trial was terminated early due to safety concerns. Interim results: patients in the 2 mg/h intravenous nimodipine group showed a significantly worse outcome at 21 days compared to the placebo. Patients in the 1 mg/h intravenous nimodipine group also showed worse outcome at 21 days but the difference was not statistically.


28 days

Within 48 h

Within 48 h

Double-blind, placebocontrolled, multicenter trial of 164 patients.

Randomized, placebocontrolled, double-blind clinical trial of 31 patients.

Placebo-controlled trial of nimodipine in the treatment of acute ischemic cerebral infarction [31] Randomized double-blind controlled study of nimodipine in acute cerebral ischemic stroke [38] 28 days

28 days

Prospective, single-center single-blind, controlled, randomized trial of 60 patients.

28 days

Nimodipine on the clinical course of patients with acute ischemic stroke [18]

Within 12 h

Randomized, doubleblind, placebocontrolled, paralleldesigned trial of 41 patients.


Nimodipine in acute ischemic stroke:a doubleblind controlled study [41]

Name of the study


120 mg/day

30 mg q.i.d. orally

120 mg/day

40 mg t.i.d.

Doses

Nimodipine was well tolerated. Mathew Scale scores showed a higher rate of improvement on nimodipine than on placebo. Mathew Scale scores revealed a highly significant difference in favor of nimodipine during the 4-week period of treatment. Mortality rates and neurologic outcome after 28 days of therapy did not differ between groups. After four weeks of treatment Mathew’s scale score improved significantly in both groups, but the difference in mean score between the two groups was insignificant.

Comments


Within 48 h

Within 48 h

Randomized, doubleblind, multicenter, placebo-controlled trial of 350 patients.

Phase III, randomized, double-blind, placebocontrolled multicenter study of 1215 patients.

TRUST. Randomized, double-blind, placebocontrolled trial of nimodipine in acute stroke [47]

21 days


Randomized, double-blind, placebo-controlled trial of nimodipine in acute ischemic hemispheric stroke [26]

Name of the study


120 mg/day orally

120 mg/day orally

Doses

There were no differences in functional outcome between the two groups. During the first month and at 3 months, there was a higher case-fatality rate in the nimodipine group than the placebo group, but at 12 months this difference had lost statistical significance. At 6 months, 55% of the nimodipine group and 58% of the placebo group were independent, the odds ratio for independence on nimodipine being 0.88 (95% confidence limits 0.70–1.10). For mortality the odds ratio with nimodipine was 1.22 (95% confidence limits 0.95–1.57).

Comments


Within 24 h

Within 24 h

Phase II, randomized, double-bind, placebocontrolled trial of 26 patients.

Flunarizine in acute ischemic stroke: a pilot study [29]

Within 6 h

Multicenter, double-blind, placebo-controlled trial of 331 patients.

Phase III, randomized, double-blind, placebocontrolled trial of 454 patients.

14 days

28 days

10 days


FIST. Flunarizine in Stroke Treatment [16]

Flunarizine

VENUS.Very Early Nimodipine Use in Stroke [24]

Name of the study


50 mg/day intravenous infusion for 7 days followed by 3 weeks of oral treatment (week 2: 21 mg/day; week 3–4: 7 mg/day). 0.1 mg/kg intravenous bolus, followed after 3 hours by continuous infusion of 0.3 mg/kg/24 hours for 3 days, followed by 10 mg/day orally for 11 days.

30 mg q.i.d. orally.

Doses

A favorable outcome on the Rankin scale was found in 32% more patients in flunarazine group than those in control group, but this difference was not statistically significant.

No differences were found between the treatment groups.

The trial was early terminated because the results did not suggest a beneficial effect of early administration of nimodipine.

Comments


Name of the study Within 12 h

Within 12 h

Phase III, randomized, multicenter, placebocontrolled trial of 1360 patients.

Phase III, multicenter, randomized trials involving 1200 patients (CLASS-I), 100–200 patients (CLASST) and 200 patients (CLASS-H).

CLASS. Clomethiazole Acute Stroke Study [49]

CLASS-IHT. Clomethiazole Acute Stroke Study in Ischemic, Hemorrhagic, and tPAtreated patients [25, 30]

Clomethiazole

1 day

1 day

68 mg/kg intravenous Infusion over 24 h.

75 mg/kg intravenous infusion over a 24h period.

Table 3 Clinical trails with drugs activating GABAA receptors Treatment: Status of the trial: clinical time from Treatment phase and number of patients onset duration Doses

There was no significant difference in functional outcome for either ischemic or hemorrhagic stroke patients. Clomethiazole frequently caused sedation. CLASS-I: No efficacy benefit was found in patients treated with clomethiazole. CLASS-H and T trials were not completed. Clomethiazole appeared to be safe but sedation was a common side effect. Development of clomethiazole has been halted due to the lack of efficacy.

Comments


Phase II safety study of ZK-200775 [15, 50]

ZK200775

ARTIST MRI. AMPA Receptor Antagonist Treatment in Ischemic Stroke [25]

ARTIST +. AMPA Receptor Antagonist Treatment in Ischemic Stroke Trial [25]

YM 872

Name of the study

Phase II, multicenter, double-blind, randomized, placebocontrolled trial of 61 patients. Dose-finding design.

Phase III, multicenter, double-blind, stratified, randomized, placebocontrolled, parallel-group trial with a planned enrollment of 600 patients. Phase II, randomized, double-blind, placebocontrolled, parallel group, multicenter trial with a planned enrollment of 260 patients.

Over 2 days

1 day

Within 6 h

Within 24 h

1 day

Within 3 h

Group 1: Total dose of 262.5 mg (i.v.) in 48 h; Group 2: Total dose of 525 mg (i.v.) in 48 h; Group 3: Total dose of 105 mg (i.v.) over a period of 6 h.

1.25 mg/kg/h intravenous infusion.

1.25 mg/kg/h intravenous infusion in combination with tPA.

Table 4 Clinical trials with drugs targeting glutamate receptors: AMPA antagonists Status of the trial: clinical Treatment: phase and number of time from Treatment patients onset duration Doses

Trial stopped prematurely due to safety concerns. ZK200775 worsened patients’ neurological condition with possible glial cell toxicity. It exerts significant sedative effects.

The trial was abandoned after failing an interim futility analysis.

The trial was abandoned after failing an interim futility analysis.

Comments


Within 6 h

Within 12 h

Phase III, randomized, multicenter, placebocontrolled, parallel-design studies in 567 patients.

Phase II, randomized, multicenter, double-blind, placebo-controlled, ascending-dose phase IIa trial of 32 patients.

ASSIST. Acute Stroke Studies Involving Selfotel Treatment [7, 8, 32]

Safety and tolerability of the glutamate antagonist CGS 19755 (selfotel) in patients with acute ischemic stroke [20]

Selfotel

Name of the study

1 day

1 day

Intravenous bolus of 1 mg/kg 2/1 day, 2 mg/kg, 1.75 mg/ kg, or 1.5 mg/kg

Single dose of 1.5 mg/ kg intravenous infusion

Trials terminated after interim analysis showed no therapeutic benefit and increase in mortality (particularly within the first 30 days and in patients with severe stroke) in selfotel group. Selfotel-treated patients had a higher incidence of agitation and hallucinations. A single intravenous dose of 1.5 mg/kg selfotel was safe and tolerable. Agitation, hallucinations, confusion, paranoia, and delirium occurred in all patients treated with 2.0 mg/kg. A significantly higher percentage of treated patients achieved an independent Barthel Index score at day 90 than placebo patients.

Table 5 Clinical trials with drugs targeting glutamate receptors: competitive NMDA antagonists Status of the trial: clinical Treatment: phase and number of time from Treatment patients onset duration Doses Comments


Safety, tolerability, and pharmacokinetics of the N-methyl-D-aspartate antagonist dextrorphan in patients with acute stroke. Dextrorphan Study Group [1]

Multicenter, ascendingdose study in 67 patients.

Within 48 h

Within 24 h

Double-blind, randomized, placebo-controlled, ascending doses, multicenter trial of 46 patients.

Safety and tolerability study of aptiganel hydrochloride in patients with an acute ischemic stroke [14]

Dextrorphan

Within 6 h

Phase II–III, multicenter, double-blind, placebocontrolled, randomized, trial of 628 patients.

Aptiganel acute stroke trial [3]

Aptiganel

Name of the study

1 day

1 day

12 h

1-h loading dose (60–150 mg) followed by a 23-h ascending-dose maintenance infusion. 1-h loading dose (145–260 mg) followed by an 11-h constant rate infusion (30–70 mg/h).

High-dose: 5-mg bolus followed by 0.75 mg/h for 12 h; Low-dose: 3-mg bolus followed by 0.5 mg/h for 12 h. 3, 4.5, 6 and 7.5 mg intravenous bolus followed by constant infusion for 6–12 h.

Loading-dose escalation was stopped because of rapid-onset, reversible, symptomatic hypotension in 7 of 21 patients treated with doses of 200–260 mg/h. There was no difference in neurological outcome at 48 h between the dextrorphan-treated and placebo-treated patients.

A 4.5-mg intravenous bolus of aptiganel followed by infusion of 0.75 mg/h for 12 was founded out a tolerable dose.

Clinical trial halted because an interim analysis showed no improvement in outcome at 90 days.

Table 6 Clinical Trials with drugs targeting glutamate receptors: NMDA channel blockers Status of the trial: clinical Treatment: phase and number of time from Treatment patients onset duration Doses Comments


Within 12 h

Within 2 h

Within 12 h

Within 12 h

Phase 2, open-label, pilot study in 20 patients.

Phase III, multicenter, randomized, doubleblind, placebocontrolled trial ongoing. Study Size Planned: 1298

Phase III, multicenter, double-blind, randomized placebocontrolled trial of 2589 patients.

Phase III, single-center, randomized, doubleblind, placebocontrolled pilot trial of 60 patients.

FAST-MAG Field Administration of Stroke Therapy – Magnesium Phase III Trial [25]

IMAGES Intravenous MAGnesium Efficacy in Stroke [37]

Randomized, double-blind, placebo-controlled pilot trial of intravenous magnesium sulfate in acute stroke [34]

1 day

1 day

1 day

1 day


FAST-MAG Pilot Study. Field Administration of Stroke TherapyMagnesium [44]

Magnesium

Name of the study

Status of the trial: clinical phase and number of patients Paramedic-administered dose of 2.5 g magnesium plus an additional 1.5 g bolus, followed by a maintenance infusion of 16 g magnesium. 4 g bolus dose of magnesium delivered over 15 min by paramedics in the field, followed by an inhospital infusion of 16 g magnesium delivered over 24 h. Intravenous magnesium sulfate in a 16-mmol bolus infused over 15 min, followed by a 65mmol maintenance dose over 24 h. 8 mmol intravenous over 15 min followed by 65 mmol over 24 h.

Doses

Magnesium was well tolerated, with no significant adverse effects and no change in blood pressure or pulse rate.

Primary outcome was not improved by intravenous magnesium sulfate.

Trial Ongoing. At this writing 651 patients have been enrolled.

Good functional outcome at 3 months occurred in 60%. Field-based magnesium intervention is safe and feasible.

Comments


Remacemide hydrochloride: a double-blind, placebocontrolled, safety and tolerability study in patients with acute ischemic stroke [12]

Remacemide

Phase Ib trial of NPS 1506 [33]

NPS 1506

Name of the study

Placebo-controlled, dose escalating, parallel group study in 61 patient.

Phase Ib, double-blind, placebo-controlled, ascending dose study in 36 patients.


Within 12 h

Within 48 h

3 days


100, 200, 300, 400, 500, or 600 mg in 6 intravenous infusions ( 2 doses per day for 3 days).

60, 80, or 100 mg, infused over 60 min.

Doses

The most common adverse events were related to the CNS, and these events appeared to increase with dose.

NPS 1506 was well tolerated at all three doses and there were no serious adverse events.

Comments


GAIN 2. Glycine Antagonist in Neuroprotection-2 [46, 25]

GAIN 1. Glycine Antagonist in Neuroprotection-1 [46, 25]

GV 150526 (Gavestinel)

Dose escalation study of the NMDA glycine-site antagonist licostinel in acute ischemic stroke [2]

ACEA 1021 (Licostinel)

Name of the study

Phase II, randomized, multicenter, doubleblind, placebocontrolled, dose escalation study on 56 patients. Phase II, randomized, multicenter, doubleblind, placebocontrolled study on 109 patients.

Dose-escalation, placebocontrolled, randomized study in 64 patients.

Within 12 h

Within 12 h

Within 48 h

3 days

3 days

1 day

800-mg loading dose followed by 200 mg every 12 h

Group 1: 400 mg; Group 2: 800 mg; Group 3: 1000 mg

Low-doses: 0.03–0.60 mg/ kg; High-doses: 1.2–3.0 mg/kg

The incidence of serious adverse events was similar in the drug and placebo groups. Hyperbilirubinemia was reported in 6% of GV150526-treated patients compared with 3% of placebo-treated patients. Outcome at 4 weeks after stroke was better in GV150526treated patients, but the

Higher doses were associated with neurological and gastrointestinal complaints. A similar improvement in National Institutes of Health Stroke Scale scores over time was seen in both the placebo group and the licostineltreated patients.

Table 7 Clinical Trials with drugs targeting glutamate receptors: NMDA glycine site antagonists Status of the trial: clinical Treatment: phase and number of time from Treatment patients onset duration Doses Comments


Glycine antagonist in neuroprotection for patients with acute Stroke: GAIN Americas: a randomized controlled trial [43] Glycine antagonist (gavestinel) in neuroprotection (GAIN International) in patients with acute stroke: a randomized controlled trial. GAIN International Investigators [27] Safety and tolerability of GV150526 (a glycine site antagonist at the Nmethyl-D-aspartate receptor) in patients with acute stroke [13]

Name of the study

Phase II, randomized, placebo-controlled, parallel-group, ascending-dose study on 66 patients and conducted in 2 phases.

Phase III, stratified, randomized, international, multicenter, doubleblind, placebo controlled trials of 1367 patients. Phase III, Multicenter, randomized, doubleblind, parallel-group, placebo-controlled trial of 1804 patients.


3 days

3 days

Within 6 h

Within 12 h

3 days

Within 6 h


Part A: loading doses of 50, 100, 200, 400, or 800 mg. Part B: loading dose of 800 mg followed by maintenance infusion.

Intravenous loading dose of 800 mg followed by 200 mg every 12 h for five doses.

Intravenous loading dose of 800 mg followed by 5 maintenance doses of 200 mg every 12 h.

Doses

GV150526 was well tolerated and no serious adverse events related to drug therapy were reported. The study was designed to assess safety and not designed to test efficacy.

There were no significant differences in serious side effects between the groups. Treatment with gavestinel within 6 h of acute ischemic stroke did not improve outcome.

studies were not powered to show statistical significance. No difference between the two group has been showed.

Comments


Multinational randomised controlled trial of lubeluzole in acute ischemic stroke. European and Australian Lubeluzole Ischemic Stroke Study Group [11] Lubeluzole in acute ischemic stroke. A double-blind, placebo-controlled phase II trial. Lubeluzole International Study Group [10]

LUB-INT-13 Lubeluzole in ischemic stroke [9]

Lubeluzole

Fosphenytoin phase 3 multicenter study to evaluate the safety and efficacy of intravenous fosphenytoin in patients with acute ischemic stroke [25, 42]

Fosphenytoin

Name of the study

Randomized, doubleblind, placebocontrolled trial of 232 patients.

Phase III international, multicenter, placebocontrolled trial of 1786 patients. Multicenter, double-blind, placebo-controlled trial of 725 patients.

Phase III, randomized, double-blind, multicenter trial of 462 patients.

5 days

5 days

Within 6 h

Within 6 h

5 days

Within 8 h

Within 4 h

Infusion of 7.5 mg over 1 h followed by 10 mg/day.

Loading infusion of 7.5 mg followed by a continuous day infusion of 10 mg. Infusion of 7.5 mg over 1 h followed by 10 mg/day.

Intravenous

Table 8 Clinical trials with drugs blocking sodium channels Status of the trial: clinical Treatment: phase and number of time from Treatment patients onset duration Doses

The trial, initially aimed at a patients inclusion of 270, was terminated prematurely because of an imbalance in mortality between the treatment group.

Treatment with lubeluzole did not affect mortality or clinical outcome in the overall study population.

No significant differences in any safety or efficacy endpoints were founded.

Enrollment halted when interim analysis found no differences between groups.

Comments


Phase II clinical trial of sipatrigine (619C89) by continuous infusion in acute stroke [36]

Phase II, randomized, placebo-controlled study of 27 patients.

Within 12 h

Within 3 h

Phase II, multicenter, placebo-controlled, randomized trial of 200 patients.

LUB-USA-6. Combination therapy with lubeluzole and t-PA in the treatment of acute ischemic stroke [21]

Sipatrigine

Within 6 h

Phase III, multicenter, randomized, doubleblind, placebocontrolled study involving 721 patients.

over 65 h

5 days

5 days


Lub. US and Canadian lubeluzole ischemic stroke study [22]

Name of the study

Status of the trial: clinical phase and number of patients Doses

10, 18, 27, or 36 mg/kg by continuous intravenous infusion.

Infusion of 7.5 mg followed by 10 mg/5 day.

7.5 mg over 1 h, followed by a continuous daily infusion of 10 mg for up to 5 days.

Comments

Neuropsychiatric effects occurred in 16 of 21 patients receiving sipatrigine. No effects on outcome measures were demonstrated.

The overall mortality rate at 12 weeks for lubeluzoletreated patients was 20.7% compared to 25.2% for placebotreated patients (Not significant). Lubeluzole treatment resulted in significantly greater improvements in functional status (Barthel Index) and overall disability (Rankin Scale) after 12 weeks. The study was halted after lack of efficacy demonstrated for lubeluzole alone.


Safety and tolerability of 619C89 after acute stroke [35]

SIS. Sipatrigine in Stroke [25]

Name of the study

Phase II trial of 170 patients. Part 1: Double-blind, placebo controlled, multicenter, escalating dose trial. Part 2: Double-blind, placebo controlled, parallel group, multicenter study. Randomized, ascendingdose, placebo-controlled trial of 48 patients.


Dose escalation was stopped after the occurrence of hallucinations in 5 of 18 patients who received 2 mg/kg/8 h or more.

Within 12 h

Comments Trial halted.

Intravenous loading dose followed by maintenance doses given 8 hourly.

Doses

Within 12 h

64 h



Post-011: Efficacy and safety of MaxiPostTM in patients with acute stroke [5]

BMS-204352

Name of the study

Phase III, randomized, Multicenter, doubleblind, placebocontrolled, parallel group study in 1978 patients. Within 6 h

Over 72 h

1 mg or 0,1 mg intravenous.

Table 9 Clinical trials with drugs opening potassium channels Status of the trial: clinical Treatment: time phase and number of from onset Treatment patients duration Doses

Study halted after negative results.

Comments



247

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Index

A Acidosis, 1, 16, 18, 117, 118, 120, 153, 154, 158, 162–164, 165, 166, 167, 168 Acid-sensing ionic channels (ASIC), vii, 3, 21, 153–168 Amiloride, 30, 73, 74, 77, 78, 120, 123, 124, 154, 158, 159, 161, 165, 166, 167 a-amino-3-hydroxy-5-methyl-4isoxazolepropionate (AMPA), 2, 4, 5, 13, 20, 21, 94, 96, 101–102, 119, 228, 236 Amlodipine, 200 Apoptosis, vi, 1, 2, 4, 5, 6, 14, 18, 36, 38, 118, 139, 140, 143, 198, 199

B BCMP1, 28, 32 Benzothiazepines, 193, 200 Blood-brain-barrier, v, 167 BMS-204353, 218, 219

C Ca2+-activated K+channels, 75, 211, 217 Ca2 cation antiporter (CaCA), 65 Ca2+/cation exchanger family (CCX), 65 CACNA1, 192, 193 Ca2+/H+exchange family (CAX), 65 Ca2+/H+exchange (YRBG), 65 Calcineurin, 53, 67, 68, 116, 117, 157, 158, 215, 217, 218 Calcium, vi, 1, 3, 15, 20, 21, 27, 29, 34, 35, 37, 38, 39, 47–58, 129, 130, 132, 136, 138, 139, 140, 142, 143, 144, 166, 189, 190, 192, 193, 198, 199, 200, 202, 228, 229 Calcium/calmodulin-dependent protein kinase II (CaMKII), 117, 157, 199

Caldendrins, 54 Calexcitin, 53 Calmodulin, 53, 116, 117, 157, 198, 199, 215 Calpains, 2, 4 Canonical (TRPC), 175 Ca2+ overload, 3, 55, 77, 100, 166, 216 Ca2 overload, 34, 39 Cariporide, 30, 39, 124 Caspases, 2, 4 Cation-chloride cotransporter superfamily (CCC), 89 CaV, 191, 192, 193, 194, 195, 196, 197, 200 Cell death, 1, 2, 3, 4, 5, 6, 7, 14, 15, 17, 19, 20, 27, 36, 38, 40, 90, 98, 101, 105, 117, 119, 121, 125, 140, 143, 175, 181, 182, 185, 198, 199 Cerebral ischemia, 1–8, 14, 16, 17, 40, 65–83, 89–106, 113–125, 153, 164, 182, 183, 184, 189, 198, 199, 201, 202 Charybdotoxin, 218 CICR, 53, 54, 55 Cl– ATPase, 7 Clot, v Conopeptides, 199 o-conotoxin, 193, 194, 199

D Dihydropyridines, 75, 190, 193, 199, 200 Diltiazem, 200

E Edema, v, 2, 5, 15, 103, 105, 118, 123, 125 Endoplasmic reticulum (ER), 4, 47–58, 130, 131, 132, 198 ER Ca2+-ATPases, 4, 75

L. Annunziato (ed.), New Strategies in Stroke Intervention, Contemporary Neuroscience, DOI 10.1007/978-1-60761-280-3, Ó Humana Press, a part of Springer ScienceþBusiness Media, LLC 2009

251

252 Exchanger inhibitory peptide (XIP), 69, 72, 75, 79 Excitotoxicity, 1, 5, 13, 14, 16, 17–20, 21, 36, 37, 95, 96, 97, 98, 101–102, 104, 123, 153

F Felodipine, 200 Flunarizine, 200, 201, 228, 234 Focal cerebral ischemia, 1, 7, 16, 17, 102

G GABA-gated Cl– channels, 7 Gabapentin, 202 Glibenclamide, 28, 36, 218, 219 GluRs, 53 Glutamate, vi, vii, 2, 3, 4, 5, 6, 7, 13–22, 36, 37, 53, 72, 94, 96, 97, 98, 99, 101, 106, 123, 162, 164, 166, 175, 181, 191, 198, 202, 211, 217, 228, 236, 237 Glutamate excitotoxicity, vi, 14, 36, 37

H Haemorrhage, v Hanatoxin, 219 HOE642, 120, 122, 124 Homeostasis, vi, vii, 1, 2, 3–7, 14, 15, 16, 47–58, 71, 75, 77, 78, 82, 90, 91, 93, 95, 100, 103, 105, 113, 118, 125, 139, 166, 180, 181, 199 Homer, 53

I Iberiotoxin, 28, 218 Infarction core, 1 Inositol-1,4,5-trisphosphate receptors (InsP3Rs), 48, 49, 50, 51, 52, 53, 54, 55, 57, 58 Inositol triphosphate (IP3), 4 Inward rectifying K+ (Kir) channels, 211 Ion homeostasis, 1, 15, 75, 103, 105 Ischemia, 15, 18, 21, 27–40, 129, 137, 140, 141, 142, 143, 144, 160, 162, 167, 168, 183, 200, 211–220, 225, 226, 227 Isradipine, 200, 201

K K+ channels, vii, 6, 8, 21, 32, 75, 159, 211–220 K+ channel openers, 6, 228

Index K+–Cl- cotransporters, 7 K+ dependent Na+/ Ca2+ exchanger (NCKXs), 3 K+-selective ion channels, vi, 211 KB-R7943, 74, 75, 78, 79, 101, 120 Kir6.1, 32 Kir6.2, 32, 36, 212–215, 216, 217, 219 Kir6.2 gene, 211–212 Kurtoxin, 199 Kv2.1, 212–215, 216, 217, 218, 219 Kv2.1 gene, 211–212

L Lamotrigine, 200, 201 Levetiracetam, 200, 201 L-type calcium channels, 198

M Melastatin (TRPM), 175 Membrane-associated guanylate kinase (MAGUK), 191 Mibefradil, 199, 200 Middle Cerebral Artery Occlusion (MCAO), 36, 67, 71, 76, 78, 106, 121, 142 Mitochondria, 2, 4, 5, 6, 27, 29, 30, 32, 33, 34, 35, 36, 37, 39, 40, 101, 102, 138, 182, 198, 214 Mitochondrial channels, 27–40 Mitochondrial dysfunction, 2, 8, 14 Mitochondrial Na+/H+ exchanger, 39 Mitochondrial Permeability Transition Pore (PTP), 34–35 MitoKAT, 28, 32, 35, 36–37, 39 MVIIA (SNX-111), 199

N Na+/Ca2+ exchanger K+-dependent family (NCKX), 65 Na+/Ca2+ exchangers (NCXs), vii, 3, 4, 21, 65, 66, 69, 70, 71, 72, 76–81, 82, 100, 101, 120, 125 NCX1, 66, 67, 69, 70, 71, 73, 74, 75, 77, 79, 80, 104 NCX2, 66, 67, 69, 70, 71, 73, 74, 75, 77, 79, 80, 82 NCX3, 66, 67–68, 69, 70, 71, 73, 74, 75, 77, 79, 80, 81 Na+/Ca2+–Li+ exchanger (NCLX or NCKX6), 65 Na+ dependent Cl-/HCO3– exchangers, 7

Index Na+/H+ exchanger (NHEs), vii, 3, 21, 113 Na+/K+-ATPase, vii, 5, 21, 75, 78, 92, 100, 124, 129–144 Na+/K+/Ca2+/Cl- cotransporters (NKCCs), vii, 3, 92 Na+-K+-Cl– cotransporter (NKCC), 89–106, 123 NAADP receptors, 48 Necrosis, vi, 5, 14, 17, 36, 38 Neurons, 2, 3, 7, 20, 37, 38, 39, 67, 68, 70, 71, 72, 81, 89, 91, 93, 94, 95, 102, 118, 119, 123, 129, 132, 140, 141, 143, 155, 156, 157, 161–162, 166, 168, 178, 179, 182, 189, 192, 194, 196, 197, 198, 199, 211, 212, 213, 214, 216, 217, 219 Neuroprotection, v, vii, 16, 18, 19, 39, 40, 97, 104, 129, 140, 141, 142, 143, 162, 164, 175, 185, 200, 201, 202, 227, 241, 242 Neurotrophins, 53 NHE, 3, 21, 73, 74, 75, 113, 118, 120, 124, 125 Nicardipine, 200, 201 Nickel, 199 Nicotinic acid adenine dinucleotide phosphate (NAADP), 48, 53 Nifedipine, 98, 199, 200 Nimodipine, 199, 200, 201, 202, 228, 229, 230, 231, 232, 233, 234 Nitric oxide, 4, 53, 136, 158 Nitric oxide synthase, 3, 164 NKCC, 21, 89, 90, 91, 106 NKCC1, 5, 89, 90, 91, 92–104, 123 NKCC2, 89, 106 NMDA, 2, 4, 13, 14, 15, 16, 18, 19, 20, 21, 22, 75, 78, 94, 96, 97, 98, 101, 102, 143, 162, 164, 165, 182, 183, 201, 228, 237, 238, 241 NMDARs, 175, 181 182, 185, 195 N-methyl-D-aspartate (NMDA), 2, 195, 238 NS1608, 218 NS1619, 28, 218 Nuclear factor-kB (NFkB), 67

253 Permanent middle cerebral artery occlusion (pMCAO), 76, 77, 80 Phenylalkylamine, 193, 200 Pimozide, 199 Plasma membrane, 3, 4, 16, 27, 28, 29, 30, 32, 65, 71, 72, 73, 89, 90, 93, 100, 113, 115, 116, 130, 131, 132, 136, 137, 139, 140, 154, 178, 191 Potassium channel openers, 6, 228 P/Q-type channels, 198, 201 Preconditioning, 6, 139 Pregabalin, 200, 202 Protein kinases, 53, 69, 117, 136, 157, 177, 199, 215 PTP (permeability transition pore), 27, 29, 34–35, 38–40

R RNS (Reactive Nitrogen Species), vi, 72, 180, 182 ROS (Reactive Oxygen Species), vi, 2, 4, 6, 27, 28, 36, 37, 38, 39, 72, 114, 180, 182 rTPA, 81 Ryanodine receptors (RyRs), 48, 49–55, 56–57

O Oxidative stress, 2, 3, 4, 5, 6, 33, 34, 92, 106 Oxygen glucose deprivation (OGD), 7, 36, 37, 39, 40, 78, 96, 100, 102, 141, 182, 199, 217

S Sabiporide, 123 SEA0400, 74, 75, 78, 79 SERCA, 48, 49, 50, 52, 54, 56, 58 SERCA (Sarco(Endo) plasmic reticulum calcium ATPase), 48 Slo1, 211–215, 216, 217, 218, 219 Slo1 gene, 211 Slotoxin, 218 SM-20220, 122, 123, 124 SN-6, 74, 75, 79 Stroke, v, vi, vii, 1, 13–22, 36–40, 47–58, 76–81, 95–104, 118–123, 129–144, 153–168, 181–183, 198–199, 216–218, 225–246 Stromatoxin, 219 Sulfonylurea receptors SUR1, 3, 32, 212, 213, 214, 215, 216, 218 SUR2, 32

P Paxilline, 28, 218 Penumbra, vi, 1, 5, 15, 16, 17, 18, 19, 78, 80, 81, 143

T TEA, 217, 218, 219, 220 Thrombolysis, v Tolbutamide, 218

254 tPA, v, 144, 181, 184, 235, 236 Transient receptor potential channels (TRPC), vii, 3, 21 Transient receptor potential (TRP), 3, 21, 176, 177, 178 Trimeric intracellular cation channels (TRICs), 48 TRPM7, 175–185 TRPV1 channels, 48 TWIK-related potassium channels (TREK), 3 TY-12533, 124 U UCPs, 27, 28, 32–34, 35, 36, 37–38, 39 UCP1, 32 UCP2, 28, 34, 38 UCP3, 34 UCP4, 28, 32, 34 UCP5, 28, 32, 34

Index V Vanilloid (TRPV), 57, 177 Verapamil, 200 Very Early Nimodipine Use in Stroke (VENUS), 201, 234 Voltage-dependent Ca2 channels (VDCC), 2, 3, 4, 7, 98 Voltage-dependent Cl- channels, 7 Voltage-gated calcium channels (VGCC), 189, 192, 193 Voltage-gated K+ (Kv) channels, 211 Voltage operated Ca2+ channels (VOCC), vii

Y YM-244769, 74, 75

Z Zinc, 15, 199

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