988 resultados para Ca2 influx
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In cardiomyocytes, calcium (Ca2+) release units comprise clusters of intracellular Ca2+ release channels located on the sarcoplasmic reticulum, and hypertension is well established as a cause of defects in calcium release unit function. Our objective was to determine whether endurance exercise training could attenuate the deleterious effects of hypertension on calcium release unit components and Ca2+ sparks in left ventricular myocytes of spontaneously hypertensive rats. Male Wistar and spontaneously hypertensive rats (4 months of age) were divided into 4 groups: normotensive (NC) and hypertensive control (HC), and normotensive (NT) and hypertensive trained (HT) animals (7 rats per group). NC and HC rats were submitted to a low-intensity treadmill running protocol (5 days/week, 1 h/day, 0% grade, and 50-60% of maximal running speed) for 8 weeks. Gene expression of the ryanodine receptor type 2 (RyR2) and FK506 binding protein (FKBP12.6) increased (270%) and decreased (88%), respectively, in HC compared to NC rats. Endurance exercise training reversed these changes by reducing RyR2 (230%) and normalizing FKBP12.6 gene expression (112%). Hypertension also increased the frequency of Ca2+ sparks (HC=7.610.26 vs NC=4.790.19 per 100 m/s) and decreased its amplitude (HC=0.2600.08 vs NC=0.3240.10 ΔF/F0), full width at half-maximum amplitude (HC=1.050.08 vs NC=1.260.01 m), total duration (HC=11.510.12 vs NC=14.970.24 ms), time to peak (HC=4.840.06 vs NC=6.310.14 ms), and time constant of decay (HC=8.680.12 vs NC=10.210.22 ms). These changes were partially reversed in HT rats (frequency of Ca2+ sparks=6.260.19 m/s, amplitude=0.2820.10 ΔF/F0, full width at half-maximum amplitude=1.140.01 m, total duration=13.340.17 ms, time to peak=5.430.08 ms, and time constant of decay=9.430.15 ms). Endurance exercise training attenuated the deleterious effects of hypertension on calcium release units of left ventricular myocytes.
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Differentes tudes ont montr que la sensibilit au Ca2+ du canal KCa3.1, un canal potassique indpendant du voltage, tait confre par la protine calmoduline (CaM) lie de faon constitutive au canal. Cette liaison impliquerait la rgion C-lobe de la CaM et un domaine de $\ikca$ directement reli au segment transmembranaire S6 du canal. La CaM pourrait galment se lier au canal de faon Ca2+ dpendante via une interaction entre un domaine de KCa3.1 du C-terminal (CaMBD2) et la rgion N-lobe de la CaM. Une tude fut entreprise afin de dterminer la nature des rsidus responsables de la liaison entre le domaine CaMBD2 de KCa3.1 et la rgion N-lobe de la CaM et leur rle dans le processus d'ouverture du canal par le Ca2+. Une structure 3D du complexe KCa3.1/CaM a d'abord t gnre par modlisation par homologie avec le logiciel MODELLER en utilisant comme rfrence la structure cristalline du complexe SK2.2/CaM (PDB: 1G4Y). Le modle ainsi obtenu de KCa3.1 plus CaM prvoit que le segment L361-S372 dans KCa3.1 devrait tre responsable de la liaison dpendante du Ca2+ du canal avec la rgion N-lobe de la CaM via les rsidus L361 et Q364 de KCa3.1 et E45, E47 et D50 de la CaM. Pour tester ce modle, les rsidus dans le segment L361-S372 ont t muts en Cys et l'action du MTSET+ (charg positivement) et MTSACE (neutre) a t mesure sur l'activit du canal. Des enregistrements en patch clamp en configuration ``inside-out`` ont montr que la liaison du ractif charg MTSET+ au le mutant Q364C entrane une forte augmentation du courant, un effet non observ avec le MTSACE. De plus les mutations E45A et E47A dans la CaM, ont empch l'augmentation du courant initi par MTSET+ sur le mutant Q364C. Une analyse en canal unitaire a confirm que la liaison MTSET+ Q364C cause une augmentation de la probabilit d'ouverture de KCa3.1 par une dstabilisation de l'tat ferm du canal. Nous concluons que nos rsultats sont compatibles avec la formation de liaisons ioniques entre les complexes chargs positivement Cys-MTSET+ la position 364 de KCa3.1 et les rsidus chargs ngativement E45 et E47 dans la CaM. Ces donnes confirment qu'une stabilisation lectrostatique des interactions CaM/KCa3.1 peut conduire une augmentation de la probabilit d'ouverture du canal en conditions de concentrations saturantes de Ca2+.
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Le diabte est une maladie chronique de lhomostasie du glucose caractrise par une hyperglycmie non contrle qui est le rsultat dune dfaillance de la scrtion dinsuline en combinaison ou non avec une altration de laction de linsuline. La surnutrition et le manque dactivit physique chez des individus qui ont des prdispositions gntiques donnent lieu la rsistance linsuline. Pendant cette priode dite de compensation o la concentration dacides gras plasmatiques est leve, lhyperinsulinmie compense pleinement pour la rsistance linsuline des tissus cibles et la glycmie est normale. Le mtabolisme du glucose par la cellule pancratique bta entrane la scrtion dinsuline. Selon le modle classique de la scrtion dinsuline induite par le glucose, laugmentation du ratio ATP/ADP rsultant de la glycolyse et de loxydation du glucose, induit la fermeture des canaux KATP-dpendant modifiant ainsi le potentiel membranaire suivi dun influx de Ca2+. Cet influx de Ca2+ permet lexocytose des granules de scrtion contenant linsuline. Plusieurs nutriments comme les acides gras sont capables de potentialiser la scrtion dinsuline. Cependant, le modle classique ne permet pas dexpliquer cette potentialisation de la scrtion dinsuline par les acides gras. Pour expliquer leffet potentialisateur des acides gras, notre laboratoire a propos un modle complmentaire o le malonyl-CoA driv du mtabolisme anaplrotique du glucose inhibe la carnitine palmitoyltransfrase-1, lenzyme qui constitue ltape limitante de loxydation des acides gras favorisant ainsi leur estrification et donc la formation de drivs lipidiques signaltiques. Le modle anaplrotique/lipidique de la scrtion d'insuline induite par le glucose prdit que le malonyl-CoA driv du mtabolisme du glucose inhibe la bta-oxydation des acides gras et augmente la disponibilit des acyl-CoA ou des acides gras non-estrifis. Les molcules lipidiques agissant comme facteurs de couplage du mtabolisme des acides gras l'exocytose d'insuline sont encore inconnus. Des travaux raliss par notre laboratoire ont dmontr quen augmentant la rpartition des acides gras vers la bta-oxydation, la scrtion dinsuline induite par le glucose tait rduite suggrant quun des drivs de lestrification des acides gras est important pour la potentialisation sur la scrtion dinsuline. En effet, des concentrations leves de glucose, les acides gras peuvent tre estrifis dabord en acide lysophosphatidique (LPA), en acide phosphatidique (PA) et en diacylglycrol (DAG) et subsquemment en triglycrides (TG). La prsente tude a tabli limportance relative du processus destrification des acides gras dans la production de facteurs potentialisant la scrtion dinsuline. Nous avions mis lhypothse que des molcules drives des processus destrification des acides gras (ex : lacide lysophosphatidique (LPA) et le diacylglycerol (DAG)) agissent comme signaux mtaboliques et sont responsables de la modulation de la scrtion dinsuline en prsence dacides gras. Afin de vrifier celle-ci, nous avons modifi le niveau dexpression des enzymes cls contrlant le processus destrification par des approches de biologie molculaire afin de changer la rpartition des acides gras dans la cellule bta. Lexpression des diffrents isoformes de la glycrol-3-phosphate acyltransfrase (GPAT), qui catalyse la premire tape destrification des acides gras a t augment et inhib. Les effets de la modulation de lexpression des isoenzymes de GPAT sur les processus destrifications, sur la bta-oxydation et sur la scrtion dinsuline induite par le glucose ont t tudis. Les diffrentes approches que nous avons utilises ont chang les niveaux de DAG et de TG sans toutefois altrer la scrtion dinsuline induite par le glucose. Ainsi, les rsultats de cette tude nont pas associ de rle pour lestrification de novo des acides gras dans leur potentialisation de la scrtion dinsuline. Cependant, lestrification des acides gras fait partie intgrante dun cycle de TG/acides gras avec sa contrepartie lipolytique. Dailleurs, des tudes parallles la mienne menes par des collgues du laboratoire ont dmontr un rle pour la lipolyse et un cycle TG/acides gras dans la potentialisation de la scrtion dinsuline par les acides gras. Paralllement nos tudes des mcanismes de la scrtion dinsuline impliquant les acides gras, notre laboratoire sintresse aussi aux effets ngatifs des acides gras sur la cellule bta. La glucolipotoxicit, rsultant dune exposition chronique aux acides gras saturs en prsence dune concentration leve de glucose, est dun intrt particulier vu la prpondrance de lobsit. Lisoforme microsomal de GPAT a aussi utilis comme outil molculaire dans le contexte de la glucolipotoxicit afin dtudier le rle de la synthse de novo de lipides complexes dans le contexte de dcompensation o la fonction des cellules bta diminue. La surexpression de lisoforme microsomal de la GPAT, menant laugmentation de lestrification des acides gras et une diminution de la bta-oxydation, nous permet de conclure que cette modification mtabolique est instrumentale dans la glucolipotoxicit.
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Les canaux calciques de type L CaV1.2 sont principalement responsables de lentree des ions calcium pendant la phase plateau du potentiel daction des cardiomyocytes ventriculaires. Cet influx calcique est requis pour initier la contraction du muscle cardiaque. Le canal CaV1.2 est un complexe oligomerique qui est compose de la sous-unite principale CaV1 et des sous-unites auxiliaires CaV et CaV21. CaV joue un role determinant dans ladressage membranaire de la sous-unite CaV1. CaV21 stabilise letat ouvert du canal mais le mecanisme moleculaire responsable de cette modulation na pas ete encore identifie. Nous avons recemment montre que cette modulation requiert une expression membranaire significative de CaV21 (Bourdin et al. 2015). CaV21 est une glycoproteine qui possede 16 sites potentiels de glycosylation de type N. Nous avons donc evalue le role de la glycosylation de type-N dans ladressage membranaire et la stabilite de CaV21. Nous avons dabord confirme que la proteine CaV21 recombinante, telle la proteine endogene, est significativement glycosylee puisque le traitement a la PNGase F se traduit par une diminution de 50 kDa de sa masse moleculaire, ce qui est compatible avec la presence de 16 sites Asn. Il sest avere par ailleurs que la mutation simultanee de 6/16 sites (6xNQ) est suffisante pour 1) reduire significativement la densite de surface de! CaV21 telle que mesuree par cytometrie en flux et par imagerie confocale 2) accelerer les cinetiques de degradation telle questimee apres arret de la synthese proteique et 3) diminuer la modulation fonctionnelle des courants generes par CaV1.2 telle quevaluee par la methode du patch-clamp . Les effets les plus importants ont toutefois ete obtenus avec les mutants N663Q, et les doubles mutants N348Q/N468Q, N348Q/N812Q, N468Q/N812Q. Ensemble, ces resultats montrent que Asn663 et a un moindre degre Asn348, Asn468 et Asn812 contribuent a la biogenese et la stabilite de CaV21 et confirment que la glycosylation de type N de CaV21 est necessaire a la fonction du canal calcique cardiaque de type L.
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Department of Biotechnology, Cochin University of Science and Technology
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Many connections in the basal ganglia are made around birth when animals are exposed to a host of new affective, cognitive, and sensori-motor stimuli. It is thought that dopamine modulates cortico-striatal synapses that result in the strengthening of those connections that lead to desired outcomes. We propose that there must be a time before which stimuli cannot be processed into functional connections, otherwise it would imply an effective link between stimulus, response, and reward in uterus. Consistent with these ideas, we present evidence that early in development dopamine neurons are electrically immature and do not produce high-frequency firing in response to salient stimuli. We ask first, what makes dopamine neurons immature? and second, what are the implications of this immaturity for the basal ganglia? As an answer to the first question, we find that at birth the outward current is small (3nS-V), insensitive to Ca2z, TEA, BK, and SK blockers. Rapidly after birth, the outward current increases to 15nS-V and becomes sensitive to Ca2z, TEA, BK, and SK blockers. We make a detailed analysis of the kinetics of the components of the outward currents and produce a model for BK and SK channels that we use to reproduce the outward current, and to infer the geometrical arrangement of BK and Ca2z channels in clusters. In the first cluster, T-type Ca2z and BK channels are coupled within distances of *20 nm (200 A). The second cluster consists of L-type Ca2z and BK channels that are spread over distances of at least 60 nm. As for the second question, we propose that early in development, the mechanism of action selection is in a locked-in state that would prevent dopamine neurons from reinforcing cortico-striatal synapses that do not have a functional experiential- based value.
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Many connections in the basal ganglia are made around birth when animals are exposed to a host of new affective, cognitive, and sensori-motor stimuli. It is thought that dopamine modulates cortico-striatal synapses that result in the strengthening of those connections that lead to desired outcomes. We propose that there must be a time before which stimuli cannot be processed into functional connections, otherwise it would imply an effective link between stimulus, response, and reward in uterus. Consistent with these ideas, we present evidence that early in development dopamine neurons are electrically immature and do not produce high-frequency firing in response to salient stimuli. We ask first, what makes dopamine neurons immature? and second, what are the implications of this immaturity for the basal ganglia? As an answer to the first question, we find that at birth the outward current is small (3nS-V), insensitive to Ca2+, TEA, BK, and SK blockers. Rapidly after birth, the outward current increases to 15nS-V and becomes sensitive to Ca2+, TEA, BK, and SK blockers. We make a detailed analysis of the kinetics of the components of the outward currents and produce a model for BK and SK channels that we use to reproduce the outward current, and to infer the geometrical arrangement of BK and Ca2+ channels in clusters. In the first cluster, T-type Ca2+ and BK channels are coupled within distances of similar to 20 nm (200 parallel to). The second cluster consists of L-type Ca2+ and BK channels that are spread over distances of at least 60 nm. As for the second question, we propose that early in development, the mechanism of action selection is in a "locked-in" state that would prevent dopamine neurons from reinforcing cortico-striatal synapses that do not have a functional experiential-based value.
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Voltage-dependent Ca2+ channels (VDCCs) have emerged as targets to treat neuropathic pain; however, amongst VDCCs, the precise role of the CaV2.3 subtype in nociception remains unproven. Here, we investigate the effects of partial sciatic nerve ligation (PSNL) on Ca2+ currents in small/medium diameter dorsal root ganglia (DRG) neurones isolated from CaV2.3(/) knock-out and wild-type (WT) mice. DRG neurones from CaV2.3(/) mice had significantly reduced sensitivity to SNX-482 versusWTmice. DRGs from CaV2.3(/) mice also had increased sensitivity to the CaV2.2 VDCC blocker -conotoxin. In WT mice, PSNL caused a significant increase in -conotoxin-sensitivity and a reduction in SNX-482-sensitivity. In CaV2.3(/) mice, PSNL caused a significant reduction in -conotoxin-sensitivity and an increase in nifedipine sensitivity. PSNL-induced changes in Ca2+ current were not accompanied by effects on voltagedependence of activation in either CaV2.3(/) or WT mice. These data suggest that CaV2.3 subunits contribute, but do not fully underlie, drug-resistant (R-type) Ca2+ current in these cells. In WT mice, PSNL caused adaptive changes in CaV2.2- and CaV2.3-mediated Ca2+ currents, supporting roles for these VDCCs in nociception during neuropathy. In CaV2.3(/) mice, PSNL-induced changes in CaV1 and CaV2.2 Ca2+ current, consistent with alternative adaptive mechanisms occurring in the absence of CaV2.3 subunits.
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Bacterial pathogens and symbionts must suppress or negate host innate immunity. However, pathogens release conserved oligomeric and polymeric molecules or MAMPs (Microbial Associated Molecular Patterns), which elicit host defenses [1], [2] and [3]. Extracellular polysaccharides (EPSs) are key virulence factors in plant and animal pathogenesis, but their precise function in establishing basic compatibility remains unclear [4], [5], [6] and [7]. Here, we show that EPSs suppress MAMP-induced signaling in plants through their polyanionic nature [4] and consequent ability to chelate divalent calcium ions [8]. In plants, Ca2+ ion influx to the cytosol from the apoplast (where bacteria multiply [4], [5] and [9]) is a prerequisite for activation of myriad defenses by MAMPs [10]. We show that EPSs from diverse plant and animal pathogens and symbionts bind calcium. EPS-defective mutants or pure MAMPs, such as the flagellin peptide flg22, elicit calcium influx, expression of host defense genes, and downstream resistance. Furthermore, EPSs, produced by wild-type strains or purified, suppress induced responses but do not block flg22-receptor binding in Arabidopsis cells. EPS production was confirmed in planta, and the amounts in bacterial biofilms greatly exceed those required for binding of apoplastic calcium. These data reveal a novel, fundamental role for bacterial EPS in disease establishment, encouraging novel control strategies.
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A large number of processes are involved in the pathogenesis of atherosclerosis but it is unclear which of them play a rate-limiting role. One way of resolving this problem is to investigate the highly non-uniform distribution of disease within the arterial system; critical steps in lesion development should be revealed by identifying arterial properties that differ between susceptible and protected sites. Although the localisation of atherosclerotic lesions has been investigated intensively over much of the 20th century, this review argues that the factor determining the distribution of human disease has only recently been identified. Recognition that the distribution changes with age has, for the first time, allowed it to be explained by variation in transport properties of the arterial wall; hitherto, this view could only be applied to experimental atherosclerosis in animals. The newly discovered transport variations which appear to play a critical role in the development of adult disease have underlying mechanisms that differ from those elucidated for the transport variations relevant to experimental atherosclerosis: they depend on endogenous NO synthesis and on blood flow. Manipulation of transport properties might have therapeutic potential. Copyright (C) 2004 S. Karger AG, Basel.
The effect of free Ca2+ on the heat stability and other characteristics of low-heat skim milk powder
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Low-heat skim milk powder (SMP), reconstituted to 25% total solids, was found to have poor heat stability. This could be improved by reducing the free Ca2+ concentration to 1.14 mm, or lower, by the addition of either Amberlite IR-120 ion-exchange resin in its sodium form or tri-sodium citrate in skim milk prior to evaporation and spray drying. Reduction in Ca2+ concentration was accompanied by increases in pH, particle size, and kinematic viscosity, and by a reduction in zeta-potential and changes in colour. In-container sterilisation of the reconstituted powder increased particle size, zeta-potential, kinematic viscosity and a* and b* values. However. Ca2+ concentration, pH and whiteness decreased. This study elucidated the importance of Ca2+ concentration and pH on heat stability of low-heat SMP, suggesting that Ca2+ concentration and pH in bulk milk are useful indicators for ensuring that spray dried milk powder has good heat stability. (C) 2009 Elsevier Ltd. All rights reserved.
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Levetiracetam (LEV) is a prominent antiepileptic drug (AED) which binds to neuronal synaptic vesicle glycoprotein 2A (SV2A) protein and has reported effects on ion channels, but retains a poorly-defined mechanism of action. Here, we investigate inhibition of voltage-dependent Ca2+ (CaV) channels as a potential mechanism by which LEV imparts effects on neuronal activity. We used electrophysiological methods to investigate the effects of LEV on cholinergic synaptic transmission and CaV channel activity in superior cervical ganglion neurons (SCGNs). In parallel, we investigated effects of the LEV inactive R-enantiomer, UCB L060. Thus, LEV, but not UCB L060 (each 100 M), inhibited synaptic transmission between SCGNs in long-term culture in a time-dependent manner, significantly reducing excitatory postsynaptic potentials (EPSP) following 30 min application. In isolated SCGNs, LEV pretreatment (1 h), but not acute (5 min) application, significantly inhibited whole-cell IBa amplitude. In current clamp recordings, LEV reduced the amplitude of the afterhyperpolarizing potential (AHP) in a Ca2+-dependent manner, but also increased action potential (AP) latency in a Ca2+-independent manner, suggesting further mechanisms associated with reduced excitability. Intracellular LEV application (4-5 min) caused a rapid inhibition of IBa amplitude to an extent comparable to that seen following extracellular LEV pretreatment ( 1 h). Neither pretreatment nor intracellular application of UCB L060 produced any inhibitory effects on IBa amplitude. These results identify a stereospecific intracellular pathway by which LEV inhibits presynaptic CaV channels; resultant reductions in neuronal excitability are proposed to contribute to the anticonvulsant effects of LEV.
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Since megakaryocytes are the cellular precursors of platelets we have investigated whether they share responses to platelet agonists, in particular collagen. Although previous studies have reported responses to thrombin in non-human megakaryocytes, through studies of single cell calcium responses and protein tyrosine-phosphorylation we demonstrate for the first time that both isolated human megakaryocytes and CD41/61-positive megakaryocytes derived in culture from CD34+ cells share responses to the platelet agonists collagen, collagen-related peptide and thrombin. The responses to either collagen or CRP were seen only in the most mature megakaryocytes and not in megakaryocyte-like cell lines, suggesting that the response to collagen is a characteristic developed late during megakaryocyte differentiation. These primary cells offer the opportunity to use many molecular and cellular techniques to study and manipulate signalling events in response to platelet receptor agonists, which cannot be performed in the small, anucleate platelet itself.
Peroxynitrite mediates disruption of Ca2+ homeostasis by carbon monoxide via Ca2+ ATPase degradation
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CO stimulates formation of NO and reactive oxygen species which, via peroxynitrite formation, inhibit Ca(2+) extrusion via PMCA, leading to disruption of Ca(2+) signaling. We propose this contributes to the neurological damage associated with CO toxicity.
