Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A mangiferina à o principal constituinte das folhas e casca do caule da Mangifera indica L. (Anacardiaceae), e possui vÃrias atividades como antioxidante, imunomodulatÃria e antiinflamatÃria. Este trabalho avaliou o efeito da mangiferina, isolada de Mangifera indica, em modelos de nocicepÃÃo quÃmica (teste de contorÃÃes abdominais induzidas por Ãcido acÃtico, teste da formalina e teste da capsaicina) e tÃrmica (teste da placa quente e teste de imersÃo da cauda) em camundongos Swiss machos. Foi investigada a participaÃÃo do sistema opiÃide, canais TRP e TRPV1, Ãxido nÃtrico e adenosina no mecanismo de aÃÃo da mangiferina. A administraÃÃo de mangiferina (30 e 100 mg/kg, v.o.) e morfina (5 mg/kg, s.c.) reduziu significativamente (p<0,05) o nÃmero de contorÃÃes abdominais induzidas pelo Ãcido acÃtico em 65, 83 e 100%, respectivamente. A naloxona antagonizou o efeito antinociceptivo da morfina e da mangiferina (30mg/kg). Assim como a morfina (5 mg/kg, s.c.), a mangiferina (100 mg/kg, v.o.) demonstrou atividade antinociceptiva nas duas fases do teste da formalina, enquanto a dose de 30 mg/kg v.o., demonstrou efeito apenas na segunda fase do teste. A naloxona antagonizou o efeito antinociceptivo da morfina e da mangiferina (100mg/kg), nas duas fases do teste. A mangiferina (10, 30 e 100 mg/kg, v.o.) e a morfina (5 mg/kg, s.c.) mostraram atividade antinociceptiva significativa no teste da caspaicina, reduzindo a nocicepÃÃo em 44, 50, 61 e 100% respectivamente. A naloxona antagonizou o efeito antinociceptivo da morfina e da mangiferina (30mg/kg) no teste da capsaicina. No modelo da capsaicina, o prÃ-tratamento com naloxonazina (30 mg/kg), vermelho de rutÃnio (3 mg/kg, s.c.), capsazepina (5 mg/kg, i.p.) e L-NAME (20 mg/kg, i.p.) demonstrou que nÃo hà envolvimento do receptor Â-opiÃide, canais TRP, canais TRPV1 e Ãxido nÃtrico, respectivamente na aÃÃo da mangiferina. O prÃ-tratamento com glibenclamida (5 mg/kg, i.p.) e 8-fenilteofilina (8 mg/kg, i.p.) apontou para a participaÃÃo de canais de potÃssio e de receptores de adenosina no mecanismo de aÃÃo da mangiferina. Nos testes da placa quente e imersÃo da cauda, a mangiferina (10, 30 e 100 mg/kg) nÃo apresentou efeito antinociceptivo. Os resultados sugerem que a mangiferina exerce atividade antinociceptiva aguda atravÃs de um mecanismo de aÃÃo perifÃrico com envolvimento do sistema opiÃide, canais de potÃssio e receptores de adenosina. Mecanismos complementares incluindo supressÃo de mediadores inflamatÃrios podem estar envolvidos. / The mangiferin is the major constituent of the leaves and bark of the Mangifera indica L. and has various activities such as antioxidant, immunomodulatory and antiinflammatory properties. This study evaluated the effect of mangiferin, isolated from M. indica, in models of chemical nociception (writhing test induced by acetic acid, formalin test and capsaicin test) and thermal (hot plate test and tail immersion test) in Swiss mice males. We investigated the involvement of the opioid system, TRPV1 and TRP channels, nitric oxide and adenosine receptors in the mechanism of action of mangiferin. Administration of mangiferin (30 and 100 mg/kg, p.o.) and morphine (5 mg/kg, s.c.) significantly reduced (p<0.05) the number of writhing induced by acetic acid in 65, 83 and 100%, respectively. Naloxone antagonized the antinociceptive effect of morphine and mangiferin (30mg/kg). Like morphine (5 mg/kg, s.c.), mangiferin (100 mg/kg, p.o.) showed antinociceptive activity in both phases of the formalin test, while the dose of 30 mg/kg p.o. showed effect only on the second test phase. Naloxone antagonized the antinociceptive effect of morphine and mangiferin (100mg/kg) in both phases of the test. The mangiferin (10, 30 and 100 mg/kg, p.o.) and morphine (5 mg/kg, s.c.) showed significant antinociceptive activity in the caspaicin test, reducing nociception at 44, 50, 61 and 100% respectively. Naloxone antagonized the antinociceptive effect of morphine and mangiferin (30mg/kg) in capsaicin test. In capsaicin test pretreatment with naloxonazine (30 mg/kg), ruthenium red (3 mg/kg, s.c.), capsazepine (5 mg/kg, i.p.) and L-NAME (20 mg/kg, i.p.) showed no involvement of &#956;-opioid receptor, TRP channels, TRPV1 channels and nitric oxide, respectively in the action of mangiferin. Pretreatment with glibenclamide (5 mg/kg, i.p.) and 8-fenilteofilina (8 mg/kg, i.p.) pointed towards the involvement of potassium channels and adenosine receptors in the mechanism of action of mangiferin. In the hot plate test and tail immersion, the mangiferin (10, 30 and 100 mg/kg) showed no antinociceptive effect. The results suggest that mangiferin exerts acute antinociceptive activity through a peripheral mechanism of action involving opioid system, potassium channels and adenosine receptors. Complementary mechanisms including suppression of inflammatory mediators may be involved.

There is a general lack of atomic resolution data of mobile regions of membrane proteins embedded in lipid bilayers. As an inherently complex system, few techniques can capture information about the mobile portions of an otherwise immobilized protein. The nature of crystallography and solid-state NMR relies on structural rigidity. Solution-state NMR relies on overall mobility of a protein for resolution. In the middle regime, there are few solutions to study these systems. The inward-rectifying, pH-gated potassium channel KcsA from Streptomyces lividans makes an excellent model for the development of methods to study mobile regions of membrane proteins. Of its 160 residues, more than a third are in extracellular do- mains and are not typically captured by solid-state NMR or crystallographic techniques. These pages present evidence that KcsA’s C-terminus is highly mobile and becomes increasingly dynamic when the protein is at low pH and high K+ concen- tration, where the channel is known to be active. By applying proton-detected, high-resolution magic angle spinning NMR (HR-MAS) to fractionally deuterated KcsA, previously unattainable correlations are collected and new resonance assignments are made, demonstrating the utility of the technique. The lipid environment is well known to regulate the function of KcsA in particular and membrane proteins in general. It is generally assumed that reconstituting KcsA into a synthetic phospholipid membranes provides the protein a well-defined environment. Data is presented here which shows that KcsA co-purifies with phosphoglycerol lipids from the E. coli membrane and that these molecules are 13C enriched in the course of isotopically labeling KcsA. Further, significant hydrolysis of both co- purifying and synthetic lipids occurs under ordinary experimental conditions. These findings demand that routine analysis of samples must include verification of the chemical integrity of lipids. Finally, the feasibility of applying dynamic nuclear polarization-enhanced NMR (DNP) to KcsA is investigated as a means of elucidating information about its termini. Although KcsA is known to enhance poorly by DNP, data presented here show that this is not an intrinsic property of the protein but rather an effect of the matrix in which KcsA is investigated. The use of a 15N-enriched free amino acid dissolved into buffers used for DNP is shown to be a powerful diagnostic internal standard.

Le maintien d'un potentiel de membrane de repos négatif est à la base de l'excitabilité neuronale. Ce potentiel négatif est généré par un courant de fuite de potassium induit par les canaux potassiques à deux domaines pore (K2P). Ils se sont révélés impliqués dans de nombreux mécanismes physiologiques et physiopathologiques tels que la dépression, la neuroprotection contre les ischémies, l'anesthésie, la migraine et la perception de la douleur. L'hétéromultimérisation est un mécanisme couramment utilisé dans la nature pour augmenter la diversité fonctionnelle des complexes protéiques. Par exemple, avec 15 gènes classés en 6 sous-familles, les canaux K2P pourraient générer 120 combinaisons et, en théorie, chacune d’elles possèderait des caractéristiques bien distinctes. Ici, nous avons d’abord étudié la capacité des membres de la même sous-famille K2P (sous-famille TREK) à s’assembler pour former des hétéromères fonctionnels dotés de nouvelles propriétés. En alliant l’optopharmacologie, une technique de précipitation de molécules uniques (SiMPull) et une technique de co-localisation à l’échelle de la molécule unique à la membrane plasmique, nous avons déterminé l’existence ainsi que la stœchiométrie des complexes créés entre TREK1, TREK2 et TRAAK. Nous avons caractérisé fonctionnellement les hétérodimères et avons constaté qu'ils formaient tous des canaux sélectifs au potassium rectifiant vers l'extérieur avec une sensibilité à la tension et aux pH variables. Ayant constaté que l’hétéromérisation est possible dans la même sous-famille, nous nous demandons si cela peut être fait entre membres de familles différentes et quelles pourraient en être les conséquences pathophysiologiques. Nous avons trouvé que TREK1 et TREK2 sont capable d’hétéromériser avec le canal plus distant TRESK, un canal K2P impliqué dans la migraine. Chez l'homme, la mutation TRESK-MT, une délétion de 2 paires de base (F139WfsX24) qui induit la formation de TRESK-MT1, un dominant négatif de TRESK, a été corrélé à la migraine. De manière surprenante, nous avons découvert que cette délétion induit un site alternatif de traduction (fsATI), menant à la formation d’un second fragment de TRESK, TRESK-MT2 qui s’assemble spécifiquement avec TREK1 et TREK2. Cet assemblage induit l’extinction des courants TREK, ce qui va augmenter l’excitabilité des neurones trijumeaux, une composante clé dans l’induction de la migraine, à l’origine du phénotype migraineux observé. Ensemble, ces résultats démontrent que l’hétéromérisation des canaux K2P n’est pas rare et doit être considérée pour comprendre leurs fonctions pathophysiologiques. Enfin, les analyses génétiques des mutations liées à des pathologies devraient désormais prendre en compte les fsATI. / Maintenance of a negative resting membrane potential underlies the basis of neuronal excitability. This negative potential is generated by a potassium leak current mediated by two-pore-domain potassium channels (K2P). Over the years, they have been shown to be involved in many physiological and pathophysiological mechanisms such as depression, neuroprotection, anesthesia, migraine and pain perception. Heteromultimerization is a mechanism commonly used to increase the functional diversity of protein complexes. For example, with 15 genes classified in 6 subfamilies, the K2P channel family can potentially generates 120 combinations and, in theory, each of them would show different functional properties. Here, we first investigated the ability of the members from the same K2P subfamily (TREK subfamily) to assemble and form functional heteromeric channels with novel properties. Using single molecule pulldown (SiMPull) from HEK cell lysates, subunit counting in the plasma membrane of living cells and opto-pharmacology, we show that the TREK channel members TREK1, TREK2, and TRAAK readily co-assemble. We functionally characterized the heterodimers and found that all combinations form outwardly rectifying potassium-selective channels but with variable voltage sensitivity and pH regulation. Having found that heteromerization is possible within the same subfamily we wonder if it can happen between members from different subfamilies with lower sequence homology and what could be the pathophysiological consequences. We found that TREK1 and TREK2 are able to heterodimerize with the distantly-related TRESK, a two-pore-domain K+ channel implicated in migraine. Notably, in humans, TRESK-MT, a 2 bp frameshift mutation (F139WfsX24), which induced the formation of TRESK-MT1 a dominant negative for TRESK, was found to perfectly segregate with typical migraine in a large pedigree. Strikingly, we found that the 2 bp frameshift mutation induced an alternative translation initiation (fsATI) which leads to the translation of a second TRESK fragment, termed TRESK-MT2. We show that by co-assembling with and inhibiting TREK1 and TREK2, TRESK-MT2 increases trigeminal sensory neuron excitability, a key component of migraine induction, leading to a migraine-like phenotype. Together these findings demonstrate that K2P heteromerization is not rare and needs to be considered to understand their pathophysiological functions and that genetic analysis of disease-related mutations should consider fsATI as a distinct class of mutations.

Les canaux ioniques sont exprimés dans tous les types cellulaires, des plantes à l’Homme, où ils sont impliqués dans de nombreux processus physiologiques. Parmi ces canaux, les canaux potassiques à deux domaines pore ou K2P forment des dimères qui produisent des courants de fond, contrôlant le potentiel de repos de la membrane et ainsi l’excitabilité cellulaire. De ce fait, ils jouent un rôle dans de nombreuses fonctions physiologiques et pathophysiologiques telles que la respiration, la nociception ou la dépression et sont de plus en plus considérés comme des cibles thérapeutiques intéressantes pour le traitement de ces pathologies. La structure de ces canaux est une caractéristique importante à considérer pour le développement de nouveaux médicaments mais les mécanismes et les régulations qui contrôlent leur activité sont peu connus.Durant mon doctorat, nous avons démontré que les canaux K2P, en particulier les sous-familles THIK et TREK, peuvent s’assembler avec un autre canal de la même sous-famille pour former un hétérodimère fonctionnel. Nous avons tout d’abord prouvé que les canaux interagissent physiquement en combinant biochimie, immunocytochimie, FRET ainsi que l’électrophysiologie. De manière intéressante, les hétérodimères ont des conductances, des régulations et une sensibilité aux agents pharmacologiques différentes de celles des canaux homodimères.Ces études montrent que la famille des canaux K2P est plus étendue et diversifiée que ce qu’il avait été attendu. La combinaison de ces canaux au sein de la même sous-famille permet de créer de nouveaux canaux fonctionnels aux propriétés originales et donc de nouvelles cibles thérapeutiques / Potassium channels are highly conserved among organisms, from plants to humans, where they are involved in several functions. Among them, the two pore domain potassium channels or K2P channels are dimers that produce background channels to control membrane resting potential and thus cell excitability. They are involved in physiological functions and diseases such as breathing, nociception or depression. They are now more and more considered as important therapeutic targets for the development of new drugs targeting these diseases. Structure-function relationship of ion channels is an important feature for the drug design but we only know little about mechanisms and regulations that control the activity of K2P channels.During my PhD, we showed that K2P channels and particularly subunits of THIK and TREK subfamilies channels can also form functional heterodimers with other subunits of the same subfamily. We first proved that subunits physically interact combining biochemistry, immunocytochemistry, FRET and electrophysiology. Interestingly, heterodimers display specific conductances, regulations and pharmacology compared to homodimers.These studies showed that the diversity and number of K2P channel conductances are larger than expected. In conclusion, mixing among subunits from the same subfamily form new channels with unique properties and so new therapeutic targets

Clinically relevant concentrations of ethanol modulate the function of a number of ion channel proteins. A fundamental question regarding the effects of alcohol is whether the drug modifies ion channels by directly binding to the protein, indirectly by perturbing the surrounding membrane lipid, or some combination of both. This thesis further characterized ethanol's site of action by examining the effects of ethanol on N-methyl-D-aspartate (NMDA) receptor/channels and large conductance Ca2+-activated K+ (BK) channels at a number of levels using direct electrophysiological methods. In Chapter One, the magnitude of ethanol's inhibition of a number of cloned heteromeric NMDA receptor/channels in the absence or presence of a number of modulators was compared. The rank order of ethanol sensitivity for the subunit combinations studied was NR1b/NR2A > NR1b/NR2B > NR1b/NR2C > NR1b/NR2D. Modulation of the receptor with Mg2+, Zn2+, the glycine antagonist 7-Chlorokynurenic Acid, or after reduction or oxidation of the redox regulatory site did not alter the ethanol sensitivity of heteromeric NMDA receptors. Therefore, the ethanol sensitivity of NMDA receptor/channels is dependent upon which NR2 subunit is present, and ethanol's site of action is unrelated to these modulatory sites on the receptor/channel protein. In Chapter Two, ethanol's site of action at cloned BK channels was characterized using of a number of 1-alkanols. Ethanol, butanol, hexanol, and heptanol reversibly and dose-dependently increased the current carried through BK channels. Longer chain 1-alkanols, such as octanol had no effect on channels. In Chapter Three, the action of ethanol on BK channels reconstituted in a number of model planar bilayers was studied. Ethanol increased the activity of BK channels incorporated in bilayers composed of phosphatidylethanolamine (PE) and phosphatidylserine (PS) or PE alone by decreasing the average amount of time channels dwelled in the closed state. There was no significant effect of alcohol on either channel conductance or unitary current. Taken together, these data suggest that ethanol action on BK channels does not require the complex membrane architecture found in native membranes, and does not require freely diffusible cytoplasmic factors or proteins.

The purpose of this work was to determine whether arachidonic acid and other fatty acids might directly regulate the behavior of ion channels. Arachidonic acid is known to be liberated from plasma membrane phospholipid upon activation of cell surface receptors, and to subsequently act as a precursor to biologically active metabolites. This study was based on the rationale that the liberated arachidonic acid itself was a potential regulator of plasma membrane ion channels. The effects of arachidonic acid and other fatty acids on the behavior of ion channels were examined in two preparations of isolated smooth muscle cells. In both cell types, K+-selective ion channels were activated both by arachidonic acid and by fatty acids that are not converted to metabolites through the cyclooxygenase and lipoxygenase metabolic pathways for arachidonic acid. These results indicate that metabolites of these pathways did not mediate the fatty acid response. Further, fatty acids were effective in cell-free patches of membrane in the absence of nucleotides and Ca++, showing that signal transduction mechanisms requiring these and other cytosolic factors were not required. Such signaling mechanisms include those involving phosphorylation, cyclic nucleotides, GTP-dependent proteins, and the NADPH-dependent cytochrome P450 metabolic pathway. Thus fatty acids themselves appear to directly activate K+ channels, much as they directly activate several enzymes, and may constitute a new class of messenger molecules acting on ion channels. The two preparations of cells used were gastric smooth muscle cells from the toad, Bufo Marinus, and pulmonary artery smooth muscle cells from the New Zealand White Rabbit. In gastric smooth muscle cells, a previously undescribed K+ channel was activated by a variety of fatty acids. This channel exhibited a conductance of approximately 50 pS, weak voltage-dependence, and K+ selectivity. The fatty acid structural features required for activation of this channel were examined by testing numerous fatty acids. Further, the same K+ channel was found to be endogenously active in the presence of Ca++ at the extracellular surface of the membrane. In pulmonary artery smooth muscle cells, fatty acids activated K+ channels of a recognizable large-conductance type that is activated by Ca++ at the intracellular membrane surface. This channel type has been widely studied but has not been reported in this preparation. Characteristic of the large-conductance, calcium-activated K+ (CAK) channel type, the channels activated by fatty acids exhibited a conductance of approximately 260 pS, strong voltage-dependence, K+ selectivity, and activation by low concentrations of Ca++ (10-7-10-6 M) at the cytosolic surface of the membrane. Lastly, these CAK channels were found to be activated by membrane stretch.

The purpose of this work was to determine whether the previously identified fatty acid activation of large conductance Ca2+-activated K+ (BK) channels from rabbit pulmonary artery smooth muscle cells was due to the direct interaction of the fatty acid with a site on the channel protein. If this was found to be the case, this study would also attempt to identify the site of fatty acid-protein interaction. Fatty acids released from membrane phospholipids by cellular phospholipases or available to the cell from the extracellular environment are important signaling molecules. Fatty acids can modulate the activity of a large number of molecules including protein kinases, phospholipases, adenylate and guanylate cyclases, G-proteins and ion channels. Fatty acids have also been shown to activate transcription of genes belonging to the steroid/thyroid superfamily of receptors. The actions of fatty acids on signal transduction pathways can be direct, whereby the fatty acid molecule itself is responsible for changes in the activity of enzymes, ion channels and other proteins. Alternatively, the effects of fatty acids may be indirect. In this case, biologically active lipids, produced from the metabolism of arachidonic acid are responsible for changes in cellular signaling. A previous study on the fatty acid modulation of rabbit pulmonary artery smooth muscle BK channel activity concluded that channel activation by fatty acids did not involve cycloxygenase, lip oxygenase and P450 metabolites (122), eliminating this indirect action of fatty acids as a possible mechanism. When dealing with the effects of fatty acids on membrane bound ion channel proteins, other mechanisms of action are also possible. For example, fatty acids are capable of entering the cell membrane and can thus affect properties of the lipid bilayer, such as membrane fluidity or membrane surface charge, that may consequently alter the activity of ion channel proteins. In addition, fatty acid mediated alterations of ion channel activity could result from the effect of fatty acids on ion channel associated proteins. To determine the mechanism of action of fatty acids on the activity of BK channels from rabbit pulmonary artery smooth muscle cells, all of the above mentioned mechanisms were considered. Most of the experiments described here were carried out using the patch-clamp technique and current recordings were performed in cell free, excised inside-out or outside-out membrane patches, in the absence of any added nucleotides and calcium. As a first step towards understanding how fatty acids modulate BK channel activity, as well as the type of protein site with which fatty acids may be interacting, we determined the structural features of the fatty acid molecule that are required for channel modulation. To do this the effects of a range of fatty acids and other lipids on BK channel activity were examined. The features required for BK channel activation were found to be the negatively charged head group and a carbon chain of greater than eight carbons. We also found that positively charged lipids produced the opposite effect of negatively charged lipids, a decrease in BK channel activity. A similar chain length requirement was also necessary for channel inhibition by positively charged lipids; short chain compounds did not alter activity while those with fourteen carbons or greater decreased activity. The identification of these required structural features suggested that a specific interaction between the charge on the lipid head group is required for channel modulation by these lipids. The requirement for a chain length of greater than eight carbons also suggests that a hydrophobic interaction is necessary for these lipids to be effective modulators of this channel. In addition, the identification of these required structural features makes it unlikely that modulation of BK channel activity by these lipid compounds is a consequence of a perturbation of the lipid environment in which the channel resides. Experiments were then carried out to determine whether modulation of BK channel activity by fatty acids and other charged lipids involved any of the following indirect mechanisms of action: 1) alterations in the concentration of calcium in the vicinity of the channel due to changes in membrane surface charge, or due to calcium stores attached to excised membrane patches, 2) alterations in the membrane electric field that the channel perceives due to changes in membrane surface charge and 3) changes in the activity of membrane bound protein kinases or protein phosphatases. In experiments where high ionic strength solutions were used to shield membrane surface charge, fatty acids and other charged lipids were still able to modulate BK channel activity suggesting that fatty acids do not act through a mechanism involving surface charge. Experiments carried out in high concentrations of EGTA (20 mM) make it unlikely that calcium is involved in the modulation of BK channels by fatty acids and other lipids. The involvement of membrane bound kinases or phosphatases is also unlikely as fatty acids effectively modulated BK channel activity in the presence of staurosporin, a kinase inhibitor, and okadaic acid, a phosphatase inhibitor. The elimination of these indirect and non-specific suggests that fatty acids and charged lipids modulate BK channel activity by directly interacting with, either the channel protein itself, or some other channel associated protein. To obtain further evidence that this indeed is the mechanism by which these lipids modulate BK channel activity; experiments were carried out to identify the site of action (i.e. side of the membrane) of both negatively and positively charged lipids. The negatively charged palmitoyl coenzyme A (PCoA) and a myristoylated positively charged peptide (myr-KPRPK), two compounds that are incapable of flipping across the bilayer, were used to identify the site of action of negatively and positively charged lipids. PCoA and myr-KPRPK produced their predicted effects of BK channel activation and suppression, respectively, only when they were applied to outside-out membrane patches. These experiments, therefore, support the contention that fatty acids and other charged lipids modulate BK channel activity by interacting with a site on the channel protein or a channel associated protein and that this site is found on the external membrane surface. If the site responsible for channel modulation by fatty acids and other charged lipids is contained within the BK channel protein itself, other members of this family may also possess this site, and thus be modulated by fatty acids. Experiments were performed, therefore, to determine whether the BK cloned channels, mslo, hslo and bslo could also be modulated by fatty acids. These cloned channels were expressed in the Xenopus oocytes, and whole-cell currents were recorded using the two-electrode voltage clamp technique. The fatty acids myristic and arachidonic acid were able to increase whole-cell current of oocytes expressing all clone types. The modulation of these cloned channels by fatty acids did not appear to involve calcium, the BK β-subunit or a bioactive metabolite of arachidonic acid. Although all possible mechanisms of action were not addressed in this study, the results support the idea that the site of fatty acid interaction resides in the channel protein itself. Taken together, therefore, these studies suggest that it is very likely that fatty acids and charged lipids modulate the activity of BK channels from smooth muscle cells of the rabbit pulmonary artery by directly interacting with an externally located site on the channel protein itself. The BK clones, mslo, hslo and bslo, are also modulated by fatty acids and it is likely that they share the same mechanism of action seen for BK channels from rabbit pulmonary artery smooth muscle cells.

Mitochondria are the major effectors of cardioprotection by procedures that open the mitochondrial ATP-sensitive potassium channel (mitoKATP), including ischemic and pharmacological preconditioning. MitoKATP opening leads to increased reactive oxygen species (ROS), which then activate a mitoKATP-associated PKCε, which phosphorylates mitoKATP and leaves it in a persistent open state (Costa, ADT and Garlid, KD. Am J Physiol 295, H874-82, 2008). Superoxide (O2•-), hydrogen peroxide (H2O2), and hydroxyl radical (HO•) have each been proposed as the signaling ROS but the identity of the ROS responsible for this feedback effect is not known. Superoxide was excluded in earlier work on the basis that it does not activate PKCε and does not induce mitoKATP opening.To further examine the identity of the signaling ROS, respiring rat heart mitochondria were preincubated with ATP and diazoxide to induce the phosphorylation-dependent open state, together with agents that may interrupt feedback activation of mitoKATP by ROS scavenging or by blocking ROS transformations. Swelling assays of the preincubated mitochondria revealed that dimethylsulfoxide (DMSO), dimethylformamide (DMF), deferoxamine, trolox, and bromoenol lactone (BEL) each blocked the ROS-dependent open state but catalase did not interfere with this step. The lack of a catalase effect and the inhibitory effects of agents acting downstream of HO• excludes H2O2 as the endogenous signaling ROS and focuses attention on HO•. In support of the hypothesis that HO• is required, we also found that HO•-scavenging by DMF blocked cardioprotection by both ischemic preconditioning and diazoxide in the Langendorff perfused rat heart. HO• itself cannot act as a signaling molecule, because its lifetime is too short and it reacts immediately with nearest neighbor phospholipids and proteins. Therefore, these findings point to a product of phospholipid peroxidation, such as hydroperoxy-fatty acids. Indeed, this hypothesis was supported by the finding that hydroperoxylinoleic acid (LAOOH) opens the ATP-inhibited mitoKATP in isolated mitochondria. This effect was blocked by the specific PKCε inhibitor peptide εV1-2, showing that LAOOH activates the mitoKATP-associated PKCε. During ischemia, catabolism of mitochondrial phospholipids is accelerated, causing accumulation of plasmalogens and free fatty acids (FA) in the heart by the action of calcium independent phospholipases A2 (iPLA2). We first assessed the role of FAs and hydroxy FAs on mitoKATP opening and cardioprotection. Swelling assays of isolated rat heart mitochondria showed that naturally formed free FAs inhibit mitoKATP opening and that they are more potent inhibitors of the pharmacological open state of mitoKATP than the phosphorylation-dependent open state. That is, sustained mitoKATP opening induced by the phosphorylation-dependent feedback loop is more resistant to FA inhibition than direct mitoKATP opening by a potassium channel opener. Moreover, rat hearts perfused with micromolar concentrations of FA were resistant to cardioprotection by diazoxide or ischemic preconditioning. Racemic bromoenol lactone (BEL), a selective inhibitor of iPLA2, confers protection to otherwise untreated Langendorff perfused hearts by preventing ischemic FA release. To bring this story full circle, BEL blocks protection afforded by preconditioning and postconditioning by preventing the iPLA2-mediated release of FAOOH generated in the conditioned heart. HO• resulting from mitoKATP opening oxidizes polyunsaturated fatty acid components of the membrane phospholipids, resulting in a peroxidized side chain. FAOOH must be released in order to act on the mitochondrial PKCε, and this is achieved by the action of iPLA2. iPLA2 is essential for most modes of cardioprotection because it catalyzes the release of FAOOH. This fully supports the hypothesis that the second messenger of cardioprotective ROS-mediated signaling is hydroperoxy fatty acid (FAOOH), a downstream oxidation product of HO•.

Myocardial ischemia, infarction, heart failure and arrhythmias are the manifestations of coronary artery disease. Reduction of ischemic damage is a major concern of cardiovascular biology research. As per recent studies, the mitochondrial ATP-sensitive potassium channel (mitoKATP) opening is believed to play key role in the physiology of cardioprotection, protection against ischemia-reperfusion injury or apoptosis. However, the structural information of mitoKATP is not precisely known. Elucidating the structural integrity and functioning of the mitoKATP is therefore a major goal of cardiovascular biology research. The known structure and function of the cell ATP-sensitive potassium channel (cellKATP) is functional in interpreting the structural and functional properties of mitoKATP. The primary goal of my research was to characterize the activity of mitoKATP in the isolated mitochondria from the control mouse heart. The mitoKATP activity, if preliminarily characterized in the control strains through the light scattering technique, then the structure of the channel could possibly be established and analyzed by means of the transgenic model and with the help of immunological techniques such as western blotting and immunoflorescence. With this experimental model it was possible to demonstrate that the mitoKATP activity in control mouse heart mitochondria is activated by potassium channel openers (KCOs) such as diazoxide and cromakalim and activators of mitoKATP such as PMA (phorbol12 myristate-13-acetate), and inhibited by KATP inhibitors such as glibenc1amide and 5-hydroxydecanoate (5 HD). It was evident that the KATP activity in mouse heart mitochondria was comparable to that exhibited by the rat heart mitochondria. The various selective and non-selective activators and inhibitors of the channel elicited their activity at a similar concentration used for the rat heart mitochondria. The results were reproducible in five independent experiments for each combination, further reinforcing the significance of existing channel activity in the mouse heart mitochondria.

Calcium tolerant rabbit cardiomyocytes, isolated by collagenase perfusion, were preincubated for varying periods of time followed by resuspension in fresh media and centrifugation into an ischaemic pellet with restricted extracellular fluid. Pellets were incubated for 240 min under oil at 37°C to mimic severe ischaemia. Time to onset of ischaemic contracture (rod to square transformation) and trypan blue permeability following resuspension in 85 mOsm media were monitored at sequential times. The protocol of Series 1 was a 5-10 min pre-incubation, immediately followed by ischaemic pelleting. Preincubation with pinacidil (50 μm) protected cells from ischaemic insult, but pinacidil added only into the ischaemic pellet did not protect. Protection was abolished by the protein kinase (PKC) inhibitors chelerythrine (10 μm) added with pinacidil and calphostin C (200nm) added only into the ischaemic pellet. Neither PKC inhibitor had an effect on injury of untreated ischaemic myocytes (data not shown). Series 2-5 were preconditioning protocols with a 10 min intervention period, followed by a 30 min oxygenated drug-free period, prior to ischaemic pelleting. In series 2 pinacidil protected cells from ischaemic insult and this protection was abolished when glyburide (10 μm) was present during preincubation, or during post-incubation and ischaemia. Glyburide only partially inhibited the protection when glyburide was added only into the ischaemic pellet. In Series 3, 8-sulfophenyltheophyline (SPT)(100 μm) or adenosine deaminase during preincubation, or SPT only added into the ischaemic pellet abolished pinacidil’s protection. In Series 4, cardiomyocytes were ischaemically preconditioned by pelleting for 10 min followed by 30 min reoxygenation. Glyburide during initial ischaemic blocked protection, but when added during post incubation and into the final pellet protection was not reduced. In Series 5 8-cyclopentyl-1,3, dipropylxanthine (DPCPX) (10 μm) added into the final pellet abolished protection by pinacidil, but not protection following ischaemic preconditioning. In contrast to pinacidil, ischaemically preconditioned cells maintain protection in the presence of glyburide, indicating that: (1) pinacidil does not exactly mimic preconditioning and (2) ischaemically preconditioned cells do not require opened K+ATP channels for protection, although they appear to be important during initiation of the preconditioned state. It is hypothesized that pinacidil opening of K+ channels may facilitate induction of preconditioning.