967 resultados para CA2 OVERLOAD
Resumo:
Purpose: Although L-type Ca2+ channels are known to play a key role in the myogenic reactivity of retinal arterial vessels, the involvement of other types of voltage-gated Ca2+ channels in this process remains unknown. In the present study we have investigated the contribution of T-type Ca2+ channels to myogenic signalling in arterioles of the rat retinal microcirculation. <br/><br/>Methods: Confocal immunolabelling of wholemount preparations was used to investigate the localisation of CaV3.1-3 channels in retinal arteriolar smooth muscle cells. T-type currents and the contribution of T-type channels to myogenic signalling were assessed by whole-cell patch-clamp recording and pressure myography of isolated retinal arteriole segments. <br/><br/>Results: Strong immunolabelling for CaV3.1 was observed on the plasma membrane of retinal arteriolar smooth muscle cells. In contrast, no expression of CaV3.2 or CaV3.3 could be detected in retinal arterioles, although these channels were present on glial cell end feet surrounding the vessels and retinal ganglion cells, respectively. TTA-A2 sensitive T-type currents were recorded in retinal arteriolar myocytes with biophysical properties distinct from those of the L-type currents present in these cells. Inhibition of T-type channels using TTA-A2 or ML-218 dilated isolated, myogenically active, retinal arterioles. <br/><br/>Conclusions: CaV3.1 T-type Ca2+ channels are functionally expressed on arteriolar smooth muscle cells of retinal arterioles and play an important role in myogenic signalling in these vessels. The work has important implications concerning our understanding of the mechanisms controlling blood flow autoregulation in the retina and its disruption during ocular disease.
Resumo:
Voltage-dependent calcium channels (VDCCs) are key elements in epileptogenesis. There are several binding-sites linked to calmodulin (CaM) and several potential CaM-dependent protein kinase II (CaMKII)-mediated phosphorylation sites in CaV1.2. The tremor rat model (TRM) exhibits absencelike seizures from 8 weeks of age. The present study was performed to detect changes in the Ca2+/CaV1.2/CaM/CaMKII pathway in TRMs and in cultured hippocampal neurons exposed to Mg2+free solution. The expression levels of CaV1.2, CaM and phosphorylated CaMKII (pCaMKII; Thr286) in these two models were examined using immunofluorescence and western blotting. Compared with Wistar rats, the expression levels of CaV1.2 and CaM were increased, and the expression of pCaMKII was decreased in the TRM hippocampus. However, the expression of the targeted proteins was reversed in the TRM temporal cortex. A significant increase in the expression of CaM and decrease in the expression of CaV1.2 were observed in the TRM cerebellum. In the cultured neuron model, pCaMKII and CaV1.2 were markedly decreased. In addition, neurons exhibiting colocalized expression of CaV1.2 and CaM immunoreactivities were detected. Furthermore, intracellular calcium concentrations were increased in these two models. For the first time, o the best of our knowledge, the data of the present study suggested that abnormal alterations in the Ca2+/CaV1.2/CaM/CaMKII pathway may be involved in epileptogenesis and in the phenotypes of TRMs and cultured hippocampal neurons exposed to Mg2+free solution.
Resumo:
Purpose<br/>The purpose of this paper is to investigate the impact of employees perceptions of high involvement work practices (HIWPs) on burnout (emotional exhaustion and depersonalisation) via the mediating role of role overload and procedural justice. Further, perceived colleague support was hypothesised to moderate the effects of role overload and procedural justice on these outcomes.<br/><br/>Design/Methodology<br/>The study was conducted on a random sample of unionised registered nurses (RNs) working in the Canadian public health care sector, stratified by mission and size of the institution to ensure representativeness. Of the 6546 nurses solicited, 2174 returned a completed questionnaire, resulting in a response rate of 33.2%. To test our hypotheses we conducted structural equation modelling (SEM) in Mplus version 6.0 (Muthen and Muthen, 1998 2010) with Maximum Likelihood (ML) estimation.<br/><br/>Results<br/>The results showed that procedural justice and role overload fully mediated the influence of HIWPs on burnout. Moreover, colleague support moderated the effects of procedural justice and role overload on emotional exhaustion but not depersonalisation.<br/><br/>Limitations<br/>The study used a cross-sectional research design and is conducted among one occupational group (i.e. nurses).<br/><br/>Research/Practical Implications<br/>The findings question the dark side of HRM in the health care context. They also contribute to the lack of theoretical and empirical work dedicated to understanding the black box problem (Castanheira and Chambel, 2010).<br/><br/>Originality/Value<br/>The study employs a well-known theoretical perspective from the occupational health psychology literature to the HR field in order to contribute to the lack of theorising in the HR-well-being link.<br/>
Resumo:
<p>Plasma membrane calmodulin-dependent calcium ATPases (PMCAs) are enzymatic systems implicated in the extrusion of calcium from the cell. We and others have previously identified molecular interactions between the cytoplasmic COOH-terminal end of PMCA and PDZ domain-containing proteins. These interactions suggested a new role for PMCA as a modulator of signal transduction pathways. The existence of other intracellular regions in the PMCA molecule prompted us to investigate the possible participation of other domains in interactions with different partner proteins. A two-hybrid screen of a human fetal heart cDNA library, using the region 652-840 of human PMCA4b (located in the catalytic, second intracellular loop) as bait, revealed a novel interaction between PMCA4b and the tumor suppressor RASSF1, a Ras effector protein involved in H-Ras-mediated apoptosis. Immunofluorescence co-localization, immunoprecipitation, and glutathione S-transferase pull-down experiments performed in mammalian cells provided further confirmation of the physical interaction between the two proteins. The interaction domain has been narrowed down to region 74-123 of RASSF1C (144-193 in RASSF1A) and 652-748 of human PMCA4b. The functionality of this interaction was demonstrated by the inhibition of the epidermal growth factor-dependent activation of the Erk pathway when PMCA4b and RASSF1 were co-expressed. This inhibition was abolished by blocking PMCA/RASSSF1 association with an excess of a green fluorescent protein fusion protein containing the region 50-123 of RASSF1C. This work describes a novel protein-protein interaction involving a domain of PMCA other than the COOH terminus. It suggests a function for PMCA4b as an organizer of macromolecular protein complexes, where PMCA4b could recruit diverse proteins through interaction with different domains. Furthermore, the functional association with RASSF1 indicates a role for PMCA4b in the modulation of Ras-mediated signaling.</p>
Resumo:
A bomba de clcio de retculo sarcoplasmtico uma das protenas mais extensivamente estudadas, capaz de interagir com vrias espcies e compostos de vandio. Combinando-se estudos de fluorescncia com ensaios cinticos de transporte e ligao de 45Ca ATPase, avaliou-se o efeito de trs complexos de vandio na funo bioenergtica e estrutural de Ca2+-ATPase. Demonstrou-se que concentraes prximas dos valores de IC50 (para a hidrlise de ATP) de BMOV-V(IV) e de PDC-V(V) no inibem significativamente a acumulao de 45Ca por sarcovesculas. Por outro lado, o complexo PDC-V(V) mostrou ser capaz de estimular a ligao de 45Ca ao retculo sarcoplasmtico, sugerindo que este complexos pode interagir com o domnio de ligao de clcio bomba. Vrios estudos de fluorescncia mostraram que BMOV-V(IV) e PDC-V(V) podero ser capazes de induzir a conformao E2 (tal como solues de decavanadato e de monovanadato) e E1 de Ca2+-ATPase (tal como solues de metavanadato), respectivamente. Apesar de o composto HAIDA-V(IV) previlegiar a conformao E1 (devido sua elevada afinidade para ies Ca2+), inibiu significativamente o transporte e a ligao de 45Ca, o que sugere que possa interagir com os locais de unio de clcio. Os resultados obtidos so consistentes com a formao de um aducto entre o composto de vandio e a protena, o que sugere um efeito na homeostasia intracelular de clcio, nos sistemas de contraco muscular e, inclusive, nas vias de aco de insulina. Cada um dos trs complexos promoveu respostas distintas na Ca2+-ATPase, sugerindo uma evidencia de actividades biolgicas diversas em funo da espcie qumica de V e do ambiente de coordenao.
Resumo:
Iron plays a central role in host-parasite interactions, since both intervenients need iron for survival and growth, but are sensitive to iron-mediated toxicity. The hosts iron overload is often associated with susceptibility to infection. However, it has been previously reported that iron overload prevented the growth of Leishmania major, an agent of cutaneous leishmaniasis, in BALB/c mice. In order to further clarify the impact of iron modulation on the growth of Leishmania in vivo, we studied the effects of iron supplementation or deprivation on the growth of L. infantum, the causative agent of Mediterranean visceral leishmaniasis, in the mouse model. We found that dietary iron deficiency did not affect the protozoan growth, whereas iron overload decreased its replication in the liver and spleen of a susceptible mouse strain. The fact that the iron-induced inhibitory effect could not be seen in mice deficient in NADPH dependent oxidase or nitric oxide synthase 2 suggests that iron eliminates L. infantum in vivo through the interaction with reactive oxygen and nitrogen species. Iron overload did not significantly alter the mouse adaptive immune response against L. infantum. Furthermore, the inhibitory action of iron towards L. infantum was also observed, in a dose dependent manner, in axenic cultures of promastigotes and amastigotes. Importantly, high iron concentrations were needed to achieve such effects. In conclusion, externally added iron synergizes with the hosts oxidative mechanisms of defense in eliminating L. infantum from mouse tissues. Additionally, the direct toxicity of iron against Leishmania suggests a potential use of this metal as a therapeutic tool or the further exploration of iron anti-parasitic mechanisms for the design of new drugs.
Resumo:
OBJECTIVE: Hereditary hemochromatosis (HH) is a disease caused by mutations in the Hfe gene characterised by systemic iron overload and associated with an increased prevalence of osteoarthritis (OA) but the role of iron overload in the development of OA is still undefined. To further understand the molecular mechanisms involved we have used a murine model of HH and studied the progression of experimental OA under mechanical stress. DESIGN: OA was surgically induced in the knee joints of 10-week-old C57BL6 (wild-type) mice and Hfe-KO mice. OA progression was assessed using histology, micro CT, gene expression and immunohistochemistry at 8 weeks after surgery. RESULTS: Hfe-KO mice showed a systemic iron overload and an increased iron accumulation in the knee synovial membrane following surgery. The histological OA score was significantly higher in the Hfe-KO mice at 8 weeks after surgery. Micro CT study of the proximal tibia revealed increased subchondral bone volume and increased trabecular thickness. Gene expression and immunohistochemical analysis showed a significant increase in the expression of matrix metallopeptidase 3 (MMP-3) in the joints of Hfe-KO mice compared with control mice at 8 weeks after surgery. CONCLUSIONS: HH was associated with an accelerated development of OA in mice. Our findings suggest that synovial iron overload has a definite role in the progression of HH-related OA
T-type Ca2+ channels, SK2 channels and SERCAs gate sleep-related oscillations in thalamic dendrites.
Resumo:
T-type Ca2+ channels (T channels) underlie rhythmic burst discharges during neuronal oscillations that are typical during sleep. However, the Ca2+-dependent effectors that are selectively regulated by T currents remain unknown. We found that, in dendrites of nucleus reticularis thalami (nRt), intracellular Ca2+ concentration increases were dominated by Ca2+ influx through T channels and shaped rhythmic bursting via competition between Ca2+-dependent small-conductance (SK)-type K+ channels and Ca2+ uptake pumps. Oscillatory bursting was initiated via selective activation of dendritically located SK2 channels, whereas Ca2+ sequestration by sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) and cumulative T channel inactivation dampened oscillations. Sk2-/- (also known as Kcnn2) mice lacked cellular oscillations, showed a greater than threefold reduction in low-frequency rhythms in the electroencephalogram of non-rapid-eye-movement sleep and had disrupted sleep. Thus, the interplay of T channels, SK2 channels and SERCAs in nRt dendrites comprises a specialized Ca2+ signaling triad to regulate oscillatory dynamics related to sleep.
Resumo:
Astrocytes are the most abundant glial cell type in the brain. Although not apposite for long-range rapid electrical communication, astrocytes share with neurons the capacity of chemical signaling via Ca(2+)-dependent transmitter exocytosis. Despite this recent finding, little is known about the specific properties of regulated secretion and vesicle recycling in astrocytes. Important differences may exist with the neuronal exocytosis, starting from the fact that stimulus-secretion coupling in astrocytes is voltage independent, mediated by G-protein-coupled receptors and the release of Ca(2+) from internal stores. Elucidating the spatiotemporal properties of astrocytic exo-endocytosis is, therefore, of primary importance for understanding the mode of communication of these cells and their role in brain signaling. We here take advantage of fluorescent tools recently developed for studying recycling of glutamatergic vesicles at synapses (Voglmaier et al., 2006; Balaji and Ryan, 2007); we combine epifluorescence and total internal reflection fluorescence imaging to investigate with unprecedented temporal and spatial resolution, the stimulus-secretion coupling underlying exo-endocytosis of glutamatergic synaptic-like microvesicles (SLMVs) in astrocytes. Our main findings indicate that (1) exo-endocytosis in astrocytes proceeds with a time course on the millisecond time scale (tau(exocytosis) = 0.24 +/- 0.017 s; tau(endocytosis) = 0.26 +/- 0.03 s) and (2) exocytosis is controlled by local Ca(2+) microdomains. We identified submicrometer cytosolic compartments delimited by endoplasmic reticulum tubuli reaching beneath the plasma membrane and containing SLMVs at which fast (time-to-peak, approximately 50 ms) Ca(2+) events occurred in precise spatial-temporal correlation with exocytic fusion events. Overall, the above characteristics of transmitter exocytosis from astrocytes support a role of this process in fast synaptic modulation.
Resumo:
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+.
Resumo:
Il y a 4 isoforme de p38 : , , , and . MK5, l'origine identifi comme tant un rgulateur de PRAK (Regulated/Activated Protein Kinase), est maintenant connu pour tre active par la protine kinase p38 (qui est un mitogne activ par la protine kinase, MAPK). Cette dernire est implique dans les mcanismes de fibrose et d'apoptose pendant l'hypertrophie cardiaque. De plus, MK5 est galement active par les MAPKs atypiques; ERK3 et ERK4. Bien quelles soient fortement exprimes dans le coeur, le rle physiologique de MK5 et ERK3 demeure inconnu. Par consquent, nous avons tudi l'effet de la constriction aortique transversale (TAC) induisant un surcharge chronique de pression chez les souris htrozygotes knockout pour MK5 (MK5+/-) ou ERK3 (ERK3+/-) et pour leurs types sauvages (MK5+/+ et ERK3+/+). Deux sem post-TAC; le ratio de poids du coeur/poids corporel a t augment chez les 2 souris MK5+/- et MK5+/+. L'chocardiographie de la trans-thoracique dmontre que la surcharge de pression a altr la fonction diastolique du ventricule gauche chez MK5+/+, mais pas chez la souris MK5+/-. De plus, nous avons observ moins de dpt de collagne, valu par une coloration au trichrome de Masson, 2 et 3 sem post-TAC chez les souris MK5+/-. Paralllement, le niveau de lARNm de collagne type1 alpha-1 a t significativement diminu dans les coeurs des souris MK5+/-, 2 et 3 sem post-TAC. De mme, ERK3, mais pas ERK5 ni p38, co-IP avec MK5 dans les 2 modles des coeurs TAC; aigus ou chroniques. En revanche, lajout exognique de GST-MK5 a abaiss ERK4 et p38, mais pas ERK3 dans les lystes de coeur de souris. Par contre, GST-ERK3 et GST-p38 ne dmontrent aucune co-IP avec MK5. Ces donnes suggrent que dans le coeur seul ERK3, et non ERK4 ou p38, est capable dinteragir avec, et rguler MK5. A niveau physiologique MK5 interagit entirement avec ERK3 et par consquent MK5 nest pas disponible pour lier les protines exogniques. Les souris htrozygotes pour ERK3 (ERK3+/-) ont galement dmontr une rduction ou une absence de collagne et une faible expression dARNm du collagne type1 alpha1, 3 sem post-TAC. Ces rsultats dmontrent un important rle pro-fibrotique de la signalisation MK5-ERK3 pendant une surcharge chronique de pression.Nous avons galement dmontr 5 variant d'pissage de (MK5.1-5), y compris la forme originale (MK5.1). MK5.2 et MK5.5 subissent une dltion de 6 paires de base dans lexon 12 : MK5.3 manque l'exon 12 : MK5.4 et MK5.5 manquent les exons 2-6. L'expression des ARNm des diffrents variant d'pissage a t vrifie par PCR en temps rel (qPCR). Bien que lexpression est ubiquitaire, l'abondance relative de chaque variant tait tissu-spcifique (coeur, rein, pancras, muscle squelettique, poumon, foie, et cerveau). En plus, l'abondance relative des variant dpissage varie pendant la surcharge de pression et le dveloppement postnatal du coeur. En outre, l'immunofluorescence a indiqu que MK5.1-5.3 se localise au noyau alors que MK5.4-5.5 est situ au niveau cytoplasmic dans les cellules HEK 293 non stimules. Suite une stimulation avec l'anisomycin, un activateur de p38 MAPK, MK5.1-5.3 se translocalise du noyau au cytoplasme alors quune petite fraction de MK5.4-5.5 translocalise vers le noyau. Ces variant d'pissage peuvent diversifier la signalisation de MK5-ERK3 dans coeur, mais leur rle exact oblige des recherches supplmentaires. Except lisoforme , toutes les isoformes de p38 sont exprimes dans le coeur et la forme est considre comme tant l'isoforme dominante. Lanalyse par qPCR et immunobuvardage de type western ont dmontr que p38 et p38 sont les deux isoformes prdominantes alors que p38 et p38 sont exprimes aux mmes niveaux dans le coeur de rat adulte. L'immunofluorescence a dmontr que p38 et p38 se trouvent dans le cytoplasme et le noyau. Cependant, suite la surcharge par TAC, p38 s'est accumul dans noyau tandis que la distribution de p38 est demeure inchange. Ainsi, l'abondance de p38 et sa translocalisation nuclaire suite la surcharge de pression indique un rle potentiel dans l'expression gnique pendant le remodelage cardiaque. En conclusion, nous avons mis en vidence pour la premire fois un rle pro-fibrotique pour la signalisation MK5-ERK3 pendant une surcharge chronique de pression. D'ailleurs, les niveaux comparables d'expression de p38 avec p38, et la localisation diffrentielle de p38 pendant la surcharge aigu ou chronique de pression suggrent diffrents rles possibles pour ces isoformes pendant le remodelage hypertrophique cardiaque.
Resumo:
Department of Biotechnology, Cochin University of Science and Technology
