Regulation of KCa2.3 andendothelium-dependent hyperpolarization (EDH) in the rat middle cerebral artery: the role of lipoxygenase metabolites and isoprostanes

Background and Purpose. In rat middle cerebral arteries, endothelium-dependent hyperpolarization (EDH) is mediated by activation of calcium-activated potassium(KCa) channels specifically KCa2.3 and KCa3.1. Lipoxygenase (LOX) products function as endothelium-derived hyperpolarizing factors (EDHFs) in rabbit arteries by stimulating KCa2.3. We investigated if LOX products contribute to EDH in rat cerebral arteries. Methods. Arachidonic acid (AA) metabolites produced in middle cerebral arteries were measured using HPLC and LC/MS. Vascular tension and membrane potential responses to SLIGRL were simultaneously recorded using wire myography and intracellular microelectrodes. Results. SLIGRL, an agonist at PAR2 receptors, caused EDH that was inhibited by a combination of KCa2.3 and KCa3.1 blockade. Non-selective LOX-inhibition reduced EDH, whereas inhibition of 12-LOX had no effect. Soluble epoxide hydrolase (sEH) inhibition enhanced the KCa2.3 component of EDH. Following NO synthase (NOS) inhibition, the KCa2.3 component of EDH was absent. Using HPLC, middle cerebral arteries metabolized 14C-AA to 15- and 12-LOX products under control conditions. With NOS inhibition, there was little change in LOX metabolites, but increased F-type isoprostanes. 8-iso-PGF2α inhibited the KCa2.3 component of EDH. Conclusions. LOX metabolites mediate EDH in rat middle cerebral arteries. Inhibition of sEH increases the KCa2.3 component of EDH. Following NOS inhibition,loss of KCa2.3 function is independent of changes in LOX production or sEH inhibition but due to increased isoprostane production and subsequent stimulation of TP receptors. These findings have important implications in diseases associated with loss of NO signaling such as stroke; where inhibition of sEH and/or isoprostane formation may of benefit.

Eag 1, Eag 2 and Kcnn3 gene brain expression of isolated reared rats

The Eag1 and Eag2, voltage-dependent potassium channels, and the small-conductance calcium-activated potassium channel (Kcnn3) are highly expressed in limbic regions of the brain, where their function is still unknown. Eag1 co-localizes with tyrosine hydroxilase enzyme in the substantia nigra and ventral tegmental area. Kcnn3 deficiency leads to enhanced serotonergic and dopaminergic neurotransmission accompanied by distinct alterations in emotional behaviors. As exposure to stress is able to change the expression and function of several ion channels, suggesting that they might be involved in the consequences of stress, we aimed at investigating Eag 1, Eag2 and Kcnn3 mRNA expression in the brains of rats submitted to isolation rearing. As the long-lasting alterations in emotional and behavioral regulation after stress have been related to changes in serotonergic neurotransmission, expressions of serotonin Htr1a and Htr2a receptors in male Wistar rats` brain were also investigated. Rats were reared in isolation or in groups of five for nine weeks after weaning. Isolated and socially reared rats were tested for exploratory activity in the open field test for 5 min and brains were processed for reverse-transcription coupled to quantitative polymerase chain reaction (qRT-PCR). Isolated reared rats showed decreased exploratory activity in the open field. Compared to socially reared rats, isolated rats showed reduced Htr2a mRNA expression in the striatum and brainstem and reduced Eag2 mRNA expression in all examined regions except cerebellum. To our knowledge, this is the first work to show that isolation rearing can change Eag2 gene expression in the brain. The involvement of this channel in stress-related behaviors is discussed.

Crown ethers : applications in inorganic synthesis

The ability of macroheterocyclic compounds to complex with ionic species has led to the synthesis and investigation of many multidentate macroheterocyclic species. The most stable complexes are formed between macrocyclic polyetheral ligands (crown ethers) with alkali or alkaline earth metal iona. There is an excellent correlation of the stability of these complexes with the size of the cation and the site of the cavity in the macrocyclic ligand. Additional factors, such as the basicity of the ligand and the solvating ability of the solvent, also play important roles in the stabilization of the complex. The stability of such complexes has been advantageously used to increase anionic reactivity and has been successfully applied to several organic fluorinations, oxidations, and similar reactions. The use of macrocyclic ligands in inorganic syntheses of otherwise difficult to obtain fluoro compounds has not been reported. O-carborane and m-carborane, C2BlOHl2, are icosahedral cage systems derived from Bl2H122- by replacement of BH with the isoelectronic CH group. These stable molecules exhibit electron-deficient bonding which can best be explained by delocalization of electrons. This delocalization gives rise to stability similar to that found in aromatic hydrocarbons. Crown ether activated potassium fluoride has been successfully employed in the conversion of alkyl, acyl and aryl halides to their respective fluorides. Analogously halide substituted carboranes were prepared, but their fluoro-derivatives were not obtained. The application of crown ethers in the synthesis of transition metal complexes is relatively unexplored. The usual synthesis of fluoro-derivative transition metal complexes involves highly reactive and toxic fluorinating agents such as antimony trifluoride, antimony penta fluoride. bromine trifluoride and hydrogen fluoride, An attempted preparation of the hexafluoroosmate (IV) ion via a crown activated, or naked fluoride~was unsuccessful. Potassium hexafluoroosmate (IV), K208F6. was eventually prepared using bromine trifluoride as a fluorinating and oxidizing agent .

Impaired beta-adrenoceptor-induced relaxation in small mesenteric arteries from DOCA-salt hypertensive rats is due to reduced K-Ca channel activity

beta-Adrenoceptor (beta-AR)-mediated relaxation plays an important role in the regulation of vascular tone. beta-AR-mediated vascular relaxation is reduced in various disease states and aging. We hypothesized that beta-AR-mediated vasodilatation is impaired in DOCA-salt hypertension due to alterations in the cAMP pathway. beta-AR-mediated relaxation was determined in small mesenteric arteries from DOCA-salt hypertensive and control uninephrectomized (Uni) rats. To exclude nitric oxide (NO) and cyclooxygenase (COX) pathways, relaxation responses were determined in the presence of L-NNA and indomethacin, NO synthase inhibitor and COX inhibitors, respectively. Isoprenaline (ISO)-induced relaxation was reduced in arteries from DOCA-salt compared to Uni rats. Protein kinase A (PKA) inhibitors (H89 or Rp-cAMPS) or adenylyl cyclase inhibitor (SQ22536) did not abolish the difference in ISO-induced relaxation between the groups. Forskolin (adenylyl cyclase activator)-induced relaxation was similar between the groups. The inhibition of IKCa/SKCa channels (TRAM-34 plus UCL1684) or BKCa channels (iberiotoxin) reduced ISO-induced relaxation only in Uni rats and abolished the relaxation differences between the groups. The expression of SKCa channel was decreased in DOCA-salt arteries. The expression of BKCa channel a subunit was increased whereas the expression of BKCa channel p subunit was decreased in DOCA-salt arteries. The expression of receptor for activated C kinase 1 (RACK1), which is a binding protein for BKG, channel and negatively modulates its activity, was increased in DOCA-salt arteries. These results suggest that the impairment of beta-AR-mediated relaxation in DOCA-salt mesenteric arteries may be attributable to altered IKCa/SKCa and/or BKCa channels activities rather than cAMP/PKA pathway. Impaired beta-AR-stimulated BKCa channel activity may be due to the imbalance between its subunit expressions and RACK1 upregulation. (C) 2012 Elsevier Ltd. All rights reserved.

Effect of chlorzoxazone in patients with downbeat nystagmus: A pilot trial

Objective: Downbeat nystagmus (DBN) is the most frequent form of acquired persisting fixation nystagmus with different symptoms such as unsteadiness of gait, postural instability, and blurred vision with reduced visual acuity (VA) and oscillopsia. However, different symptomatic therapeutic principles are required, such as 3,4-diaminopyridine and 4-aminopyridine, that effectively suppress DBN. Chlorzoxazone (CHZ) is a nonselective activator of small conductance calcium-activated potassium (SK) channels that modifies the activity of cerebellar Purkinje cells. We evaluated the effects of this agent on DBN in an observational proof-of-concept pilot study. Methods: Ten patients received CHZ 500 mg 3 times a day for 1 or 2 weeks. Slow-phase velocity of DBN, VA, postural sway, and the drug's side effects were evaluated. Recordings were conducted at baseline, 90 minutes after first administration, and after 1 or 2 weeks. Results: Mean slow-phase velocity significantly decreased from a baseline of 2.74°/s ± 2.00 to 2.29°/s ± 2.12 (mean ± SD) 90 minutes after first administration and to 2.04°/s ± 2.24 (p < 0.001; post hoc both p = 0.024) after long-term treatment. VA significantly increased and postural sway in posturography showed a tendency to decrease on medication. Fifty percent of patients did not report any side effects. The most common reported side effect was abdominal discomfort and dizziness. Conclusions: The treatment with the SK-channel activator CHZ is a potentially new therapeutic agent for the symptomatic treatment of DBN. Classification of evidence: This study provides Class IV evidence that CHZ 500 mg 3 times a day may improve eye movements and visual fixation in patients with DBN.

Detection of synchrony in the activity of auditory nerve fibers by octopus cells of the mammalian cochlear nucleus

The anatomical and biophysical specializations of octopus cells allow them to detect the coincident firing of groups of auditory nerve fibers and to convey the precise timing of that coincidence to their targets. Octopus cells occupy a sharply defined region of the most caudal and dorsal part of the mammalian ventral cochlear nucleus. The dendrites of octopus cells cross the bundle of auditory nerve fibers just proximal to where the fibers leave the ventral and enter the dorsal cochlear nucleus, each octopus cell spanning about one-third of the tonotopic array. Octopus cells are excited by auditory nerve fibers through the activation of rapid, calcium-permeable, α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptors. Synaptic responses are shaped by the unusual biophysical characteristics of octopus cells. Octopus cells have very low input resistances (about 7 MΩ), and short time constants (about 200 μsec) as a consequence of the activation at rest of a hyperpolarization-activated mixed-cation conductance and a low-threshold, depolarization-activated potassium conductance. The low input resistance causes rapid synaptic currents to generate rapid and small synaptic potentials. Summation of small synaptic potentials from many fibers is required to bring an octopus cell to threshold. Not only does the low input resistance make individual excitatory postsynaptic potentials brief so that they must be generated within 1 msec to sum but also the voltage-sensitive conductances of octopus cells prevent firing if the activation of auditory nerve inputs is not sufficiently synchronous and depolarization is not sufficiently rapid. In vivo in cats, octopus cells can fire rapidly and respond with exceptionally well-timed action potentials to periodic, broadband sounds such as clicks. Thus both the anatomical specializations and the biophysical specializations make octopus cells detectors of the coincident firing of their auditory nerve fiber inputs.

Tertiapin-Q blocks recombinant and native large conductance K+ channels in a use-dependent manner

Tertiapin, a short peptide from honey bee venom, has been reported to specifically block the inwardly rectifying K+ (Kir) channels, including G protein-coupled inwardly rectifying potassium channel (GIRK) 1 + GIRK4 heteromultimers and ROMK1 homomultimers. In the present study, the effects of a stable and functionally similar derivative of tertiapin, tertiapin-Q, were examined on recombinant human voltage-dependent Ca2+-activated large conductance K+ channel (BK or MaxiK; alpha-subunit or hSlo1 homomultimers) and mouse inwardly rectifying GIRK1 + GIRK2 (i.e., Kir3.1 and Kir3.2) heteromultimeric K+ channels expressed in Xenopus oocytes and in cultured newborn mouse dorsal root ganglion (DRG) neurons. In two-electrode voltage-clamped oocytes, tertiapin-Q (1-100 nM) inhibited BK-type K+ channels in a use- and concentration-dependent manner. We also confirmed the inhibition of recombinant GIRK1 + GIRK2 heteromultimers by tertiapin-Q, which had no effect on endogenous depolarization- and hyperpolarization-activated currents sensitive to extracellular divalent cations (Ca2+, Mg2+, Zn2+, and Ba2+) in defolliculated oocytes. In voltage-clamped DRG neurons, tertiapin-Q voltage- and use-dependently inhibited outwardly rectifying K+ currents, but Cs+-blocked hyperpolarization-activated inward currents including I-H were insensitive to tertiapin-Q, baclofen, barium, and zinc, suggesting absence of functional GIRK channels in the newborn. Under current-clamp conditions, tertiapin-Q blocked the action potential after hyperpolarization (AHP) and increased action potential duration in DRG neurons. Taken together, these results demonstrate that the blocking actions of tertiapin-Q are not specific to Kir channels and that the blockade of recombinant BK channels and native neuronal AHP currents is use-dependent. Inhibition of specific types of Kir and voltage-dependent Ca2+-activated K+ channels by tertiapin-Q at nanomolar range via different mechanisms may have implications in pain physiology and therapy.

SK channels regulate excitatory synaptic transmission and plasticity in the lateral amygdala

At glutamatergic synapses, calcium influx through NMDA receptors (NMDARs) is required for long-term potentiation (LTP); this is a proposed cellular mechanism underlying memory and learning. Here we show that in lateral amygdala pyramidal neurons, SK channels are also activated by calcium influx through synaptically activated NMDARs, resulting in depression of the synaptic potential. Thus, blockade of SK channels by apamin potentiates fast glutamatergic synaptic potentials. This potentiation is blocked by the NMDAR antagonist AP5 (D(-)-2-amino-5-phosphono-valeric acid) or by buffering cytosolic calcium with BAPTA. Blockade of SK channels greatly enhances LTP of cortical inputs to lateral amygdala pyramidal neurons. These results show that NMDARs and SK channels are colocalized at glutamatergic synapses in the lateral amygdala. Calcium influx through NMDARs activates SK channels and shunts the resultant excitatory postsynaptic potential. These results demonstrate a new role for SK channels as postsynaptic regulators of synaptic efficacy.

Theoretical investigations of intercellular communication in the microcirculation: Conducted responses, myoendothelial projections and endothelium derived hyperpolarizing factor

The contractile state of microcirculatory vessels is a major determinant of the blood pressure of the whole systemic circulation. Continuous bi-directional communication exists between the endothelial cells (ECs) and smooth muscle cells (SMCs) that regulates calcium (Ca2+) dynamics in these cells. This study presents theoretical approaches to understand some of the important and currently unresolved microcirculatory phenomena. ^ Agonist induced events at local sites have been shown to spread long distances in the microcirculation. We have developed a multicellular computational model by integrating detailed single EC and SMC models with gap junction and nitric oxide (NO) coupling to understand the mechanisms behind this effect. Simulations suggest that spreading vasodilation mainly occurs through Ca 2+ independent passive conduction of hyperpolarization in RMAs. Model predicts a superior role for intercellular diffusion of inositol (1,4,5)-trisphosphate (IP3) than Ca2+ in modulating the spreading response. ^ Endothelial derived signals are initiated even during vasoconstriction of stimulated SMCs by the movement of Ca2+ and/or IP3 into the EC which provide hyperpolarizing feedback to SMCs to counter the ongoing constriction. Myoendothelial projections (MPs) present in the ECs have been recently proposed to play a role in myoendothelial feedback. We have developed two models using compartmental and 2D finite element methods to examine the role of these MPs by adding a sub compartment in the EC to simulate MP with localization of intermediate conductance calcium activated potassium channels (IKCa) and IP3 receptors (IP 3R). Both models predicted IP3 mediated high Ca2+ gradients in the MP after SMC stimulation with limited global spread. This Ca 2+ transient generated a hyperpolarizing feedback of ∼ 2–3mV. ^ Endothelium derived hyperpolarizing factor (EDHF) is the dominant form of endothelial control of SMC constriction in the microcirculation. A number of factors have been proposed for the role of EDHF but no single pathway is agreed upon. We have examined the potential of myoendothelial gap junctions (MEGJs) and potassium (K+) accumulation as EDHF using two models (compartmental and 2D finite element). An extra compartment is added in SMC to simulate micro domains (MD) which have NaKα2 isoform sodium potassium pumps. Simulations predict that MEGJ coupling is much stronger in producing EDHF than alone K+ accumulation. On the contrary, K+ accumulation can alter other important parameters (EC V m, IKCa current) and inhibit its own release as well as EDHF conduction via MEGJs. The models developed in this study are essential building blocks for future models and provide important insights to the current understanding of myoendothelial feedback and EDHF.^

Catalytic conversion to N2O to N2 over potassium catalyst supported on activated carbon

Catalytic conversion of N2O to N-2 With potassium catalysts supported on activated carbon (K/AC) was investigated. Potassium proves to be much more active and stable than either copper or cobalt because potassium possesses strong abilities both for N2O chemisorption and oxygen transfer. Potassium redispersion is found to play a critical role in influencing the catalyst stability. A detailed study of the reaction mechanism was conducted based upon three different catalyst loadings. It was found that during temperature-programmed reaction (TPR), the negative oxygen balance at low temperatures (< 50 degrees C) is due to the oxidation of the external surface of potassium oxide particles, while the bulk oxidation accounts for the oxygen accumulation at higher temperatures (below ca. 270 degrees C). N2O is beneficial for the removal of carbon-oxygen complexes because of the formation of CO2 instead of CO and because of its role in making the chemisorption of produced CO2 on potassium oxide particles less stable. A conceptual three-zone model was proposed to clarify the reaction mechanism over K/AC catalysts. CO2 chemisorption at 250 degrees C proves to be an effective measurement of potassium dispersion. (C) 1999 Academic Press.

Phosphorylation of the voltage-gated potassium channel Kv2.1 by AMP-activated protein kinase regulates membrane excitability

Firing of action potentials in excitable cells accelerates ATP turnover. The voltage-gated potassium channel Kv2.1 regulates action potential frequency in central neurons, whereas the ubiquitous cellular energy sensor AMP-activated protein kinase (AMPK) is activated by ATP depletion and protects cells by switching off energy-consuming processes. We show that treatment of HEK293 cells expressing Kv2.1 with the AMPK activator A-769662 caused hyperpolarizing shifts in the current-voltage relationship for channel activation and inactivation. We identified two sites (S440 and S537) directly phosphorylated on Kv2.1 by AMPK and, using phosphospecific antibodies and quantitative mass spectrometry, show that phosphorylation of both sites increased in A-769662-treated cells. Effects of A-769662 were abolished in cells expressing Kv2.1 with S440A but not with S537A substitutions, suggesting that phosphorylation of S440 was responsible for these effects. Identical shifts in voltage gating were observed after introducing into cells, via the patch pipette, recombinant AMPK rendered active but phosphatase-resistant by thiophosphorylation. Ionomycin caused changes in Kv2.1 gating very similar to those caused by A-769662 but acted via a different mechanism involving Kv2.1 dephosphorylation. In cultured rat hippocampal neurons, A-769662 caused hyperpolarizing shifts in voltage gating similar to those in HEK293 cells, effects that were abolished by intracellular dialysis with Kv2.1 antibodies. When active thiophosphorylated AMPK was introduced into cultured neurons via the patch pipette, a progressive, time-dependent decrease in the frequency of evoked action potentials was observed. Our results suggest that activation of AMPK in neurons during conditions of metabolic stress exerts a protective role by reducing neuronal excitability and thus conserving energy.

Photolytic manipulation of [Ca2+](i) reveals slow kinetics of potassium channels underlying the afterhyperpolarization in hipppocampal pyramidal neurons

The identity of the potassium channel underlying the slow, apamin-insensitive component of the afterhyperpolarization current (sl(AHP)) remains unknown. We studied sl(AHP) in CA1 pyramidal neurons using simultaneous whole-cell recording, calcium fluorescence imaging, and flash photolysis of caged compounds. Intracellular calcium concentration ([Ca2+](i)) peaked earlier and decayed more rapidly than sl(AHP). Loading cells with low concentrations of the calcium chelator EGTA slowed the activation and decay of sl(AHP). In the presence of EGTA, intracellular calcium decayed with two time constants. When [Ca2+](i) was increased rapidly after photolysis of DM-Nitrophen, both apamin-sensitive and apamin-insensitive outward currents were activated. The apamin-sensitive current activated rapidly (<20 msec), whereas the apamin-insensitive current activated more slowly (180 msec). The apamin-insensitive current was reduced by application of serotonin and carbachol, confirming that it was caused by sl(AHP) channels. When [Ca2+](i) was decreased rapidly via photolysis of diazo-2, the decay of sl(AHP) was similar to control (1.7 sec). All results could be reproduced by a model potassium channel gated by calcium, suggesting that the channels underlying sl(AHP) have intrinsically slow kinetics because of their high affinity for calcium.

Developmental changes in hyperpolarization-activated currents I-h and I-K(IR) in isolated rat intracardiac neurons

The hyperpolarization-activated nonselective cation current, I-h, was investigated in neonatal and adult rat intracardiac neurons. I-h was observed in all neurons studied and displayed slow time-dependent rectification. I-h was isolated by blockade with external Cs+ (2 mM) and was inhibited irreversibly by the bradycardic agent, ZD 7288. Current density of I-h was approximately twofold greater in neurons from neonatal (-4.1 pA/pF at -130 mV) as compared with adult (-2.3 pA/pF) rats; however, the reversal potential and activation parameters were unchanged. The reversal potential and amplitude of I-h was sensitive to changes in external Na+ and K+ concentrations. An inwardly rectifying K+ current, I-K(IR), was also present in intracardiac neurons from adult but not neonatal rats and was blocked by extracellular Ba2+. I-K(IR) was present in approximately one-third of the adult intracardiac neurons studied, with a current density of -0.6 pA/pF at -130 mV. I-K(IR) displayed rapid activation kinetics and no time-dependent rectification consistent with the rapidly activating, inward K+ rectifier described in other mammalian autonomic neurons. I-K(IR) was sensitive to changes in external K+, whereby raising the external K+ concentration from 3 to 15 mM shifted the reversal potential by approximately +36 mV. Substitution of external Na+ had no effect on the reversal potential or amplitude of I-K(IR). I-K(IR) density increases as a function of postnatal development in a population of rat intracardiac neurons, which together with a concomitant decrease in I-h may contribute to changes in the modulation of neuronal excitability in adult versus neonatal rat intracardiac ganglia.

The effect of water inorganic matrix in ibuprofen adsorption onto activated carbon for water and wastewater treatment

Dissertação para obtenção do Grau de Mestre em Engenharia Química e Bioquímica

Characterisation of "fou2", a constitutive wound-activated mutant and analysis of the proteome and leaf physiology in the region proximal to wounds in Arabidopsis

Résumé : Les jasmonates (JA), une famille d'hor1none végétale, jouent un rôle central dans la réponse à la blessure, et aux attaques d'insectes et de pathogènes. Les JA sont principalement dérivés d'un acide gras, l'acide linolénique. L'addition par une lipoxygénase d'une molécule d'oxygène à l'acide linolénique initie la synthèse de JA. Cependant les mécanismes régulant l'activation de la biosynthèse de JA ne sont pas encore connus. C'est pour cette raison que dans ce travail, nous avons caractérisé chez Arabidopsis thaliana (l'Arabette des Dames) un mutant fou2 dont l'activité lipoxygénase est plus élevée que celle d'une plante sauvage. Les niveaux de JA sont constitutivement plus élevés et l'activation de la synthèse de JA après blessure est fortement plus induite chez fou2 que chez le type sauvage. En outre, fou2 est plus résistant au pathogène Botrytis cinerea et à la chenille Spodoptera littoralis. Afin de comprendre quel mécanisme chez fou2 génére ce phénotype, nous avons cloné le gène responsable du phénotype de fou2. Le mutant fou2 porte une mutation dans le gène d'un canal à deux pores transportant probablement du potassium, du lumen de la vacuole végétale vers le compartiment cytosolique. L'analyse du protéome de fou2 a permis d'identifier une expression plus élevée de sept protéines régulées par les JA ou le stress. La découverte de l'implication d'un canal dans le phénotype de fou2 renforce l'hypothèse que les flux de cations pourraient être impliqués dans les étapes précoces de la synthèse des JA. Nous avons également étudié le protéome et la physiologie d'une feuille blessée, Pour évaluer les changements d'expression protéique en réponse à la blessure et contrôlés par les JA, nous avons quantifié l'expression de 5937 protéines chez une plante d'Arabidopsis sauvage et chez un mutant incapable de synthétiser des JA. Parmi ces 5937 protéines, nous avons identifié 99 protéines régulées par la blessure chez le type sauvage. Nous avons observé pour 65% des protéines dont l'expression protéique changeait après blessure une bonne corrélation entre la quantité de transcrits et de protéines. Plusieurs enzymes de la voie des chorismates impliquées dans la biosynthèse des acides aminés phénoliques étaient induites par les JA après blessure. Une quantification des acides aminés a montré que les niveaux d'acides aminés phénoliques augmentaient significativement après blessure. La blessure induisait aussi des changements dans l'expression de protéines impliquées dans la réponse au stress et particulièrement au stress oxydatif. Nous avons quantifié l'état réduit et oxydé du glutathion, un tripeptide qui, sous sa forme réduite, est l'antioxydant majeur des cellules. Nous avons trouvé une quantité significativement plus élevée de glutathion oxydé chez le type sauvage blessé que chez la plante aus blessée. Ce résultat suggère que la génération d'un stress oxydatif et la proportion relative de glutathions réduits et oxydés sont contrôlés par les JA après blessure. Abstract : Plants possess a family of potent fatty acid-derived wound-response and developmental regulators: the jasmonates. These compounds are derived from the tri?unsaturated fatty acid a-linolenic-acid (18:3). Addition of an oxygen molecule to 18:3 by 13-lipoxygenases (13-LOX) initiates JA biosynthesis. Actually components regulating the activation of JA biosynthesis are poorly defined. Therefore we characterized in Arabidopsis thaliana the fatty acid Qxygenation upregulated 2 (fou2) mutant, which was previously isolated in a screen for mutants with an enhanced 13-LOX activity. As a consequence of this increased 13-LOX activity, JA levels in fou2 are higher than in wild type (WT) and wounding strongly increased JA biosynthesis compared to WT. fou2 was more resistant to the fungus Botrytis cinerea and the generalist caterpillar Spodaptera littomlis, The fou2 mutant carries a missense mutation in the Two Pore Channel 1 gene (TPCJ), which encodes a vacuolar cation channel transporting probably K* into the cytosol. Patchclamp analysis of fou2 vacuolar membranes showed faster time-dependent conductivity and activation of the mutated channel at lower membrane potentials than wild-type. Proteomic analysis of fou2 leaves identified increased levels of seven biotic stress- and JA- inducible proteins. The discovery of the implication of a channel in the fou2 phenotype strenghtens the hypothesis that cation fluxes might be implicated in early steps of JA synthesis. We further concentrated on the proteome and leaf physiology in the region proximal to wounds in Arabidopsis using the WT and the aos JA-biosynthesis deficient mutant in order to find JA- induced proteins changes. We used two successive proteomic methods to assess protein changes in response to wounding Arabidopsis leaves, two dimensional electrophoresis (2DE) and linear trap quadrupole ion-trap mass spectrometry. In total 5937 proteins were quantified. We identified 99 wound-regulated proteins in the WT. Most these proteins were also wound-regulated at the transcript level showing a good correlation between transcript and protein abundance. We identified several wound-regulated enzymes involved in amino acid biosynthesis and confirmed this result by amino acid quantification. Proteins involved in stress reponses were upregulated, particularly in redox species regulation. We found a significantly higher quantity of oxidized glutathione in wounded WT relative to wounded aos leaves. This result suggests that levels of reduced glutathione are controlled by JA after wounding.