Étude de l'oligomérisation et de la fonction de canaux ioniques par spectroscopie de fluorescence et fluorométrie en voltage imposé

La fonction des canaux ioniques est finement régulée par des changements structuraux de sites clés contrôlant l’ouverture du pore. Ces modulations structurales découlent de l’interaction du canal avec l’environnement local, puisque certains domaines peuvent être suffisamment sensibles à des propriétés physico-chimiques spécifiques. Les mouvements engendrés dans la structure sont notamment perceptibles fonctionnellement lorsque le canal ouvre un passage à certains ions, générant ainsi un courant ionique mesurable selon le potentiel électrochimique. Une description détaillée de ces relations structure-fonction est cependant difficile à obtenir à partir de mesures sur des ensembles de canaux identiques, puisque les fluctuations et les distributions de différentes propriétés individuelles demeurent cachées dans une moyenne. Pour distinguer ces propriétés, des mesures à l’échelle de la molécule unique sont nécessaires. Le but principal de la présente thèse est d’étudier la structure et les mécanismes moléculaires de canaux ioniques par mesures de spectroscopie de fluorescence à l’échelle de la molécule unique. Les études sont particulièrement dirigées vers le développement de nouvelles méthodes ou leur amélioration. Une classe de toxine formeuse de pores a servi de premier modèle d’étude. La fluorescence à l’échelle de la molécule unique a aussi été utilisée pour l’étude d’un récepteur glutamate, d’un récepteur à la glycine et d’un canal potassique procaryote. Le premier volet porte sur l’étude de la stœchiométrie par mesures de photoblanchiment en temps résolu. Cette méthode permet de déterminer directement le nombre de monomères fluorescents dans un complexe isolé par le décompte des sauts discrets de fluorescence suivant les événements de photoblanchiment. Nous présentons ici la première description, à notre connaissance, de l’assemblage dynamique d’une protéine membranaire dans un environnement lipidique. La toxine monomérique purifiée Cry1Aa s’assemble à d’autres monomères selon la concentration et sature en conformation tétramérique. Un programme automatique est ensuite développé pour déterminer la stœchiométrie de protéines membranaires fusionnées à GFP et exprimées à la surface de cellules mammifères. Bien que ce système d’expression soit approprié pour l’étude de protéines d’origine mammifère, le bruit de fluorescence y est particulièrement important et augmente significativement le risque d’erreur dans le décompte manuel des monomères fluorescents. La méthode présentée permet une analyse rapide et automatique basée sur des critères fixes. L’algorithme chargé d’effectuer le décompte des monomères fluorescents a été optimisé à partir de simulations et ajuste ses paramètres de détection automatiquement selon la trace de fluorescence. La composition de deux canaux ioniques a été vérifiée avec succès par ce programme. Finalement, la fluorescence à l’échelle de la molécule unique est mesurée conjointement au courant ionique de canaux potassiques KcsA avec un système de fluorométrie en voltage imposé. Ces enregistrements combinés permettent de décrire la fonction de canaux ioniques simultanément à leur position et densité alors qu’ils diffusent dans une membrane lipidique dont la composition est choisie. Nous avons observé le regroupement de canaux KcsA pour différentes compositions lipidiques. Ce regroupement ne paraît pas être causé par des interactions protéine-protéine, mais plutôt par des microdomaines induits par la forme des canaux reconstitués dans la membrane. Il semble que des canaux regroupés puissent ensuite devenir couplés, se traduisant en ouvertures et fermetures simultanées où les niveaux de conductance sont un multiple de la conductance « normale » d’un canal isolé. De plus, contrairement à ce qui est actuellement suggéré, KcsA ne requiert pas de phospholipide chargé négativement pour sa fonction. Plusieurs mesures indiquent plutôt que des lipides de forme conique dans la phase cristalline liquide sont suffisants pour permettre l’ouverture de canaux KcsA isolés. Des canaux regroupés peuvent quant à eux surmonter la barrière d’énergie pour s’ouvrir de manière coopérative dans des lipides non chargés de forme cylindrique.

Conception et évaluation d’un nouveau système de transfection ciblée, basé sur l’utilisation du système E/Kcoil

Actuellement le polyéthylènimine (PEI) est l’agent de transfection transitoire le plus utilisé par l’industrie pharmaceutique pour la production de protéines recombinantes à grande échelle par les cellules de mammifères. Il permet la condensation de l’ADN plasmidique (ADNp) en formant spontanément des nanoparticules positives appelées polyplexes, lui procurant la possibilité de s’attacher sur la membrane cellulaire afin d’être internalisé, ainsi qu’une protection face aux nucléases intracellulaires. Cependant, alors que les polyplexes s’attachent sur la quasi-totalité des cellules seulement 5 à 10 % de l’ADNp internalisé atteint leur noyau, ce qui indique que la majorité des polyplexes ne participent pas à l’expression du transgène. Ceci contraste avec l’efficacité des vecteurs viraux où une seule particule virale par cellule peut être suffisante. Les virus ont évolués afin d’exploiter les voies d’internalisation et de routage cellulaire pour exprimer efficacement leur matériel génétique. Nous avons donc supposé que l’exploitation des voies d’internalisation et de routage cellulaire d’un récepteur pourrait, de façon similaire à plusieurs virus, permettre d’optimiser le processus de transfection en réduisant les quantités d’ADNp et d’agent de transfection nécessaires. Une alternative au PEI pour transfecter les cellules de mammifèreest l’utilisation de protéines possédant un domaine de liaison à l’ADNp. Toutefois, leur utilisation reste marginale à cause de la grande quantité requise pour atteindre l’expression du transgène. Dans cette étude, nous avons utilisé le système E/Kcoil afin de cibler un récepteur membranaire dans le but de délivrer l’ADNp dans des cellules de mammifères. Le Ecoil et le Kcoil sont des heptapeptides répétés qui peuvent interagir ensemble avec une grande affinité et spécificité afin de former des structures coiled-coil. Nous avons fusionné le Ecoil avec des protéines capables d’interagir avec l’ADNp et le Kcoil avec un récepteur membranaire que nous avons surexprimé dans les cellules HEK293 de manière stable. Nous avons découvert que la réduction de la sulfatation de la surface cellulaire permettait l’attachement ciblé sur les cellules par l’intermédiaire du système E/Kcoil. Nous démontrons dans cette étude comment utiliser le système E/Kcoil et une protéine interagissant avec l’ADNp pour délivrer un transgène de manière ciblée. Cette nouvelle méthode de transfection permet de réduire les quantités de protéines nécessaires pour l’expression du transgène.

Amino acids substitutions in σ1 and μ1 outer capsid proteins of a Vero cell-adapted mammalian orthoreovirus are required for optimal virus binding and disassembly

In a recent study, the serotype 3 Dearing strain of mammalian orthoreovirus was adapted to Vero cells; cells that exhibit a limited ability to support the early steps of reovirus uncoating and are unable to produce interferon as an antiviral response upon infection. The Vero cell-adapted virus (VeroAV) exhibits amino acids substitutions in both the σ1 and μ1 outer capsid proteins but no changes in the σ3 protein. Accordingly, the virus was shown not to behave as a classical uncoating mutant. In the present study, an increased ability of the virus to bind at the Vero cell surface was observed and is likely associated with an increased ability to bind onto cell-surface sialic acid residues. In addition, the kinetics of μ1 disassembly from the virions appears to be altered. The plasmid-based reverse genetics approach confirmed the importance of σ1 amino acids substitutions in VeroAV's ability to efficiently infect Vero cells, although μ1 co-adaptation appears necessary to optimize viral infection. This approach of combining in vitro selection of reoviruses with reverse genetics to identify pertinent amino acids substitutions appears promising in the context of eventual reovirus modification to increase its potential as an oncolytic virus.

L’importance de la coopération de TRAF1 et LSP1 en aval du récepteur 4-1BB(CD137) pour la survie des lymphocytes

4-1BB (CD137) est un membre de la superfamille TNFR qui est impliqué dans la transmission des signaux de survie aux lymphocytes. TRAF1 est une protéine adaptatrice qui est recrutée par 4-1BB et autres TNFRs et est caractérisée par une expression très restreinte aux lymphocytes, cellules dendritiques et certaines cellules épithéliales. TRAF1 est nécessaire pour l’expansion et la survie des cellules T mémoire en présence d'agonistes anti-4-1BB in vivo. De plus, TRAF1 est requise en aval de 4-1BB pour activer (phosphoryler) la MAP kinase Erk impliquée dans la régulation de la molécule pro-apoptotique Bim. Suite à l’activation du récepteur 4-1BB, TRAF1 et ERK sont impliqués dans la phosphorylation de Bim et la modulation de son expression. L’activation et la régulation de TRAF1 et Bim ont un rôle important dans la survie des cellules T CD8 mémoires. Dans cette étude, nous avons utilisé une approche protéomique afin de pouvoir identifier de nouveaux partenaires de liaison de TRAF1. Utilisant cette stratégie, nous avons identifié que LSP1 (Leukocyte Specific Protein 1) est recruté dans le complexe de signalisation 4-1BB de manière TRAF1 dépendante. Une caractérisation plus poussée de l’interaction entre TRAF1 et LSP1 a montré que LSP1 lie la région unique N-terminal de TRAF1 de façon indépendante de la région conservée C-terminal. À l’instar des cellules T déficientes en TRAF1, les cellules T déficientes en LSP1 ne sont pas capables d’activer ERK en aval de 4-1BB et par conséquent ne peuvent pas réguler Bim. Ainsi, TRAF1 et LSP1 coopèrent en aval de 4-1BB dans le but d’activer ERK et réguler en aval les niveaux de Bim dans les cellules T CD8. Selon la littérature, le récepteur 4-1BB n’est pas exprimé à la surface des cellules B murines, mais le récepteur 4-1BB favorise la prolifération et la survie des cellules B humaines. Cependant, il est important d'étudier l'expression du récepteur 4-1BB dans les cellules B murines afin de disposer d'un modèle murin et de prédire la réponse clinique à la manipulation de 4-1BB. En utilisant différentes stimulations de cellules B murines primaires, nous avons identifié que le récepteur 4-1BB est exprimé à la surface des cellules B de souris suite à une stimulation avec le LPS (Lipopolysaccharides). Une caractérisation plus poussée a montré que le récepteur 4-1BB est induit dans les cellules B murines d'une manière dépendante de TLR4 (Toll Like Receptor 4). Collectivement, notre travail a démontré que la stimulation avec le LPS induit l’expression du récepteur 4-1BB à la surface des cellules B murines, menant ainsi à l'induction de TRAF1. De plus, TRAF1 et LSP1 coopèrent en aval de 4-1BB pour activer la signalisation de la Map kinase ERK dans les cellules B murines de manière similaire aux cellules T. Les cellules B déficientes en TRAF1 et les cellules B déficientes en LSP1 ne sont pas en mesure d'activer la voie ERK en aval de 4-1BB et montrent un niveau d’expression du récepteur significativement diminué comparé aux cellules B d’une souris WT. Ainsi, TRAF1 et LSP1 sont nécessaires pour une expression maximale du récepteur 4-1BB à la surface cellulaire de cellules B murines et coopèrent en aval de 4-1BB afin d'activer la cascade ERK dans les cellules B murines.

Neurotransmitters Functional Balance in Neurodegenerative Disease Management:recent Advances

The recent developments in neurobiology have rendered new prominence and potential to study about the structure and function of brain and related disorders. Human behaviour is the net result of neural control of the communication between brain cells. Neurotransmitters are chemicals that are used to relay, amplify and modulate electrical signals between neurons and/or another cell. It mediates rapid intercellular communication through the nervous system by interacting with cell surface receptors. These receptors often trigger second messenger signaling pathways that regulate the activity of ion channels. The functional balance of different neurotransmitters such as Acetylcholine (Ach), Dopamine (DA), Serotonin (5-HT), Norepinephrine (NE), Epinephrine (EPI), Glutamate and Gamma amino butyric acid (GABA) regulates the growth, division and other vital functions of a normal cell / organism (Sudha, 1998). Any change in neurotransmitters' functional balance will result in the failure of cell function and may lead to the occurrence of diseases. Abnormalities in the production or functioning of neurotransmitters have been implicated in a number of neurological disorders like Schizophrenia, Alzheimer's, Epilepsy, Depression and Parkinson's disease. Changes in central and peripheral neuronal signaling system is also noted in diabetes, cancer, cell proliferation, alcoholism and aging. Elucidation of neurotransmitters receptor interaction pathways and gene expression regulation by second messengers and transcriptional factors in health and disease conditions can lead to new small molecules for development of therapeutic agents to improve neurological disease conditions. Increased awareness of the global effects of neurological disorders should help health care planners and the neurological community set appropriate priorities in research, prevention, and management of these diseases.

Interaction of severe acute respiratory syndrome-coronavirus and NL63 coronavirus spike proteins with angiotensin converting enzyme-2

Although in different groups, the coronaviruses severe acute respiratory syndrome-coronavirus (SARS-CoV) and NL63 use the same receptor, angiotensin converting enzyme (ACE)-2, for entry into the host cell. Despite this common receptor, the consequence of entry is very different; severe respiratory distress in the case of SARS-CoV but frequently only a mild respiratory infection for NL63. Using a wholly recombinant system, we have investigated the ability of each virus receptor-binding protein, spike or S protein, to bind to ACE-2 in solution and on the cell surface. In both assays, we find that the NL63 S protein has a weaker interaction with ACE-2 than the SARS-CoV S protein, particularly in solution binding, but the residues required for contact are similar. We also confirm that the ACE-2-binding site of NL63 S lies between residues 190 and 739. A lower-affinity interaction with ACE-2 might partly explain the different pathological consequences of infection by SARS-CoV and NL63.

Platelet PECAM-1 inhibits thrombus formation in vivo

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a cell surface glycoprotein receptor expressed on a range of blood cells, including platelets, and on vascular endothelial cells. PECAM-1 possesses adhesive and signaling properties, the latter being mediated by immunoreceptor tyrosine-based inhibitory motifs present on the cytoplasmic tail of the protein. Recent studies in vitro have demonstrated that PECAM-1 signaling inhibits the aggregation of platelets. In the present study we have used PECAM-1-deficient mice and radiation chimeras to investigate the function of this receptor in the regulation of thrombus formation. Using intravital microscopy and laser-induced injury to cremaster muscle arterioles, we show that thrombi formed in PECAM-1-deficient mice were larger, formed more rapidly than in control mice, and were more stable. Larger thrombi were also formed in control mice that received transplants of PECAM-1-deficient bone marrow, in comparison to mice that received control transplants. A ferric chloride model of thrombosis was used to investigate thrombus formation in carotid arteries. In PECAM-1-deficient mice the time to 75% vessel occlusion was significantly shorter than in control mice. These data provide evidence for the involvement of platelet PECAM-1 in the negative regulation of thrombus formation.

Extracellular disulfide exchange and the regulation of cellular function

An emerging concept is that disulfide bonds can act as a dynamic scaffold to present mature proteins in different conformational and functional states on the cell surface. Two examples are the conversion of the receptor, integrin a alpha(IIb)beta(3), from a low affinity to a high affinity state, and the interaction of CD4 receptor with the HIV-1 envelope glycoprotein gp120 to promote virus-cell fusion. In both of these cases there is a remodeling of the protein disulfide bonding pattern. The formation and rearrangement of disulfide bonds is modulated by a family of enzymes known as the thiol isomerases, which include protein disulfide isomerase (PDI), ERp5, ERp57, and ERp72. While these enzymes were reported originally to be restricted in location to the endoplasmic reticulum, in some cells thiol isomerases are found on the cell surface. This may indicate a wider role for these enzymes in cell function. In platelets it has been shown that reagents that react with cell surface sulfhydryl groups are capable of blocking a number of functional responses, including integrin-mediated aggregation, adhesion, and granule secretion. Furthermore, the use of function blocking antibodies to either PDI or ERp5 causes inhibition of these functional responses. This review summarizes current knowledge of the extracellular regulation of disulfide exchange and the implications of this in the regulation of cell function.

Infection of human airway epithelium by human and avian strains of influenza a virus

We describe the characterization of influenza A virus infection of an established in vitro model of human pseudostratified mucociliary airway epithelium (HAE). Sialic acid receptors for both human and avian viruses, alpha-2,6- and alpha-2,3-linked sialic acids, respectively, were detected on the HAE cell surface, and their distribution accurately reflected that in human tracheobronchial tissue. Nonciliated cells present a higher proportion of alpha-2,6-linked sialic acid, while ciliated cells possess both sialic acid linkages. Although we found that human influenza viruses infected both ciliated and nonciliated cell types in the first round of infection, recent human H3N2 viruses infected a higher proportion of nonciliated cells in HAE than a 1968 pandemic-era human virus, which infected proportionally more ciliated cells. In contrast, avian influenza viruses exclusively infected ciliated cells. Although a broad-range neuraminidase abolished infection of HAE by human parainfluenza virus type 3, this treatment did not significantly affect infection by influenza viruses. All human viruses replicated efficiently in HAE, leading to accumulation of nascent virus released from the apical surface between 6 and 24 h postinfection with a low multiplicity of infection. Avian influenza A viruses also infected HAE, but spread was limited compared to that of human viruses. The nonciliated cell tropism of recent human H3N2 viruses reflects a preference for the sialic acid linkages displayed on these cell types and suggests a drift in the receptor binding phenotype of the H3 hemagglutinin protein as it evolves in humans away from its avian virus precursor.

A role for the thiol isomerase. protein ERP5 in platelet function

Formation and rearrangement of disulfide bonds during the correct folding of nascent proteins is modulated by a family of enzymes known as thiol isomerases, which include protein disulfide isomerase (PDI), endoplasmic reticulum protein 5 (ERP5), and ERP57. Recent evidence supports an alternative role for this family of proteins on the surface of cells, where they are involved in receptor 'remodeling and recognition. In platelets, blocking PDI with inhibitory antibodies inhibits a number of platelet activation pathways, including aggregation, secretion, and fibrinogen binding. Analysis of human platelet membrane fractions identified the presence of the thiol isomerase protein ERP5. Further study showed that ERP5 is resident mainly on platelet intracellular membranes, although it is rapidly recruited to the cell, surface in response to a range of platelet agonists. Blocking cell-surface ERP5 using inhibitory antibodies leads to a decrease in platelet aggregation in response to agonists, and a decrease in fibrinogen binding and P-selectin exposure. It is Possible that this is based on the disruption of integrin function, as we observed that ERP5 becomes physically associated with the integrin beta(3) subunit during platelet stimulation. These results provide new insights into the involvement of thiol isomerases and regulation of platelet activation. (C) 2005 by The American Society of Hematology.

Protein-disulfide isomerase-mediated reduction of two disulfide bonds of HIV envelope glycoprotein 120 occurs post-CXCR4 binding and is required for fusion

The human immunodeficiency virus (HIV) envelope (Env) glycoprotein (gp) 120 is a highly disulfide-bonded molecule that attaches HIV to the lymphocyte surface receptors CD4 and CXCR4. Conformation changes within gp120 result from binding and trigger HIV/cell fusion. Inhibition of lymphocyte surface-associated protein-disulfide isomerase (PDI) blocks HIV/cell fusion, suggesting that redox changes within Env are required. Using a sensitive assay based on a thiol reagent, we show that (i) the thiol content of gp120, either secreted by mammalian cells or bound to a lymphocyte surface enabling CD4 but not CXCR4 binding, was 0.5-1 pmol SH/pmol gp120 (SH/gp120), whereas that of gp120 after its interaction with a surface enabling both CD4 and CXCR4 binding was raised to 4 SH/gp120; (ii) PDI inhibitors prevented this change; and (iii) gp120 displaying 2 SH/gp120 exhibited CD4 but not CXCR4 binding capacity. In addition, PDI inhibition did not impair gp120 binding to receptors. We conclude that on average two of the nine disulfides of gp120 are reduced during interaction with the lymphocyte surface after CXCR4 binding prior to fusion and that cell surface PDI catalyzes this process. Disulfide bond restructuring within Env may constitute the molecular basis of the post-receptor binding conformational changes that induce fusion competence.

Synthesis and structure-activity relationship studies of CD4 down-modulating cyclotriazadisulfonamide (CADA) analogues

HIV attachment via the CD4 receptor is an important target for developing novel approaches to HIV chemotherapy. Cyclotriazadisulfonamide (CADA) inhibits HIV at submicromolar levels by specifically down-modulating cell-surface and intracellular CD4. An effective five-step synthesis of CADA in 30% overall yield is reported. This synthesis has also been modified to produce more than 50 analogues. Many tail-group analogues have been made by removing the benzyl tail of CADA and replacing it with various alkyl, acyl, alkoxycarbonyl and aminocarbonyl substituents. A series of sidearm analogues, including two unsymmetrical compounds, have also been prepared by modifying the CADA synthesis, replacing the toluenesulfonyl sidearms with other sulfonyl groups. Testing 30 of these compounds in MT-4 cells shows a wide range of CD4 down-modulation potency, which correlates with ability to inhibit HIV-1. Three-dimensional quantitative structure-activity relationship (3D-QSAR) models were constructed using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) approaches. The X-ray crystal structures of four compounds, including CADA, show the same major conformation of the central 12-membered ring. The solid-state structure of CADA was energy minimized and used to generate the remaining 29 structures, which were similarly minimized and aligned to produce the 3D-QSAR models. Both models indicate that steric bulk of the tail group, and, to a lesser extent, the sidearms mainly determine CD4 down-modulation potency in this series of compounds.

Mathematical model for low density lipoprotein (LDL) endocytosis by hepatocytes

Individuals with elevated levels of plasma low density lipoprotein (LDL) cholesterol (LDL-C) are considered to be at risk of developing coronary heart disease. LDL particles are removed from the blood by a process known as receptor-mediated endocytosis, which occurs mainly in the liver. A series of classical experiments delineated the major steps in the endocytotic process; apolipoprotein B-100 present on LDL particles binds to a specific receptor (LDL receptor, LDL-R) in specialized areas of the cell surface called clathrin-coated pits. The pit comprising the LDL-LDL-R complex is internalized forming a cytoplasmic endosome. Fusion of the endosome with a lysosome leads to degradation of the LDL into its constituent parts (that is, cholesterol, fatty acids, and amino acids), which are released for reuse by the cell, or are excreted. In this paper, we formulate a mathematical model of LDL endocytosis, consisting of a system of ordinary differential equations. We validate our model against existing in vitro experimental data, and we use it to explore differences in system behavior when a single bolus of extracellular LDL is supplied to cells, compared to when a continuous supply of LDL particles is available. Whereas the former situation is common to in vitro experimental systems, the latter better reflects the in vivo situation. We use asymptotic analysis and numerical simulations to study the longtime behavior of model solutions. The implications of model-derived insights for experimental design are discussed.

Mathematical modelling of competitive LDL/VLDL binding and uptake by hepatocytes

Elevated levels of low-density-lipoprotein cholesterol (LDL-C) in the plasma are a well-established risk factor for the development of coronary heart disease. Plasma LDL-C levels are in part determined by the rate at which LDL particles are removed from the bloodstream by hepatic uptake. The uptake of LDL by mammalian liver cells occurs mainly via receptor-mediated endocytosis, a process which entails the binding of these particles to specific receptors in specialised areas of the cell surface, the subsequent internalization of the receptor-lipoprotein complex, and ultimately the degradation and release of the ingested lipoproteins' constituent parts. We formulate a mathematical model to study the binding and internalization (endocytosis) of LDL and VLDL particles by hepatocytes in culture. The system of ordinary differential equations, which includes a cholesterol-dependent pit production term representing feedback regulation of surface receptors in response to intracellular cholesterol levels, is analysed using numerical simulations and steady-state analysis. Our numerical results show good agreement with in vitro experimental data describing LDL uptake by cultured hepatocytes following delivery of a single bolus of lipoprotein. Our model is adapted in order to reflect the in vivo situation, in which lipoproteins are continuously delivered to the hepatocyte. In this case, our model suggests that the competition between the LDL and VLDL particles for binding to the pits on the cell surface affects the intracellular cholesterol concentration. In particular, we predict that when there is continuous delivery of low levels of lipoproteins to the cell surface, more VLDL than LDL occupies the pit, since VLDL are better competitors for receptor binding. VLDL have a cholesterol content comparable to LDL particles; however, due to the larger size of VLDL, one pit-bound VLDL particle blocks binding of several LDLs, and there is a resultant drop in the intracellular cholesterol level. When there is continuous delivery of lipoprotein at high levels to the hepatocytes, VLDL particles still out-compete LDL particles for receptor binding, and consequently more VLDL than LDL particles occupy the pit. Although the maximum intracellular cholesterol level is similar for high and low levels of lipoprotein delivery, the maximum is reached more rapidly when the lipoprotein delivery rates are high. The implications of these results for the design of in vitro experiments is discussed.

Modulation of interleukin signalling and gene expression in cardiac myocytes by endothelin-1

The related inflammatory cytokines, interleukin- (IL-) 1β and IL-33, are both implicated in the response of the heart to injury. They also activate mitogen-activated protein kinases (MAPKs) in cardiac myocytes. The hypertrophic Gq protein-coupled receptor agonist endothelin-1 is a potentially cardioprotective peptide and may modulate the inflammatory response. Endothelin-1 also stimulates (MAPKs) in cardiac myocytes and promotes rapid changes in expression of mRNAs encoding intercellular and intracellular signalling components including receptors for IL-33 (ST2) and phosphoprotein phosphatases. Prior exposure to endothelin-1 may specifically modulate the response to IL-33 and, more globally, influence MAPK activation by different stimuli. Neonatal rat ventricular myocytes were exposed to IL-1β or IL-33 with or without pre-exposure to endothelin-1 (5 h) and MAPK activation assessed. IL-33 activated ERK1/2, JNKs and p38-MAPK, but to a lesser degree than IL-1β. Endothelin-1 increased expression of soluble IL-33 receptors (sST2 receptors) which may prevent binding of IL-33 to the cell-surface receptors. However, pretreatment with endothelin-1 only inhibited activation of p38-MAPK by IL-33 with no significant influence on ERK1/2 and a small increase in activation of JNKs. Inhibition of p38-MAPK signalling following pretreatment with endothelin-1 was also detected with IL-1β, H2O2 or tumour necrosis factor α (TNFα) indicating an effect intrinsic to the signalling pathway. Endothelin-1 pretreatment suppressed the increase in expression of IL-6 mRNA induced by IL-1β and decreased the duration of expression of TNFα mRNA. Coupled with the general decrease in p38-MAPK signalling, we conclude that endothelin-1 attenuates the cardiac myocyte inflammatory response, potentially to confer cardioprotection.