Calcineurin

Distribution by Scientific Domains
Distribution within Life Sciences

Kinds of Calcineurin

  • phosphatase calcineurin

  • Terms modified by Calcineurin

  • calcineurin activity
  • calcineurin inhibition
  • calcineurin inhibitor
  • calcineurin inhibitor toxicity

  • Selected Abstracts


    Inactivation of calcineurin by hydrogen peroxide and phenylarsine oxide

    FEBS JOURNAL, Issue 5 2000
    Evidence for a dithiol, disulfide equilibrium, implications for redox regulation
    Calcineurin (CaN) is a Ca2+ -and calmodulin (CaM)-dependent serine/threonine phosphatase containing a dinuclear Fe,Zn center in the active site. Recent studies have indicated that CaN is a possible candidate for redox regulation. The inactivation of bovine brain CaN and of the catalytic CaN A-subunit from Dictyostelium by the vicinal dithiol reagents phenylarsine oxide (PAO) and melarsen oxide (MEL) and by H2O2 was investigated. PAO and MEL inhibited CaN with an IC50 of 3,8 µm and the inactivation was reversed by 2,3-dimercapto-1-propane sulfonic acid. The treatment of isolated CaN with hydrogen peroxide resulted in a concentration-dependent inactivation. Analysis of the free thiol content performed on the H2O2 inactivated enzyme demonstrated that only two or three of the 14 Cys residues in CaN are modified. The inactivation of CaN by H2O2 could be reversed with 1,4-dithiothreitol and with the dithiol oxidoreductase thioredoxin. We propose that a bridging of two closely spaced Cys residues in the catalytic CaN A-subunit by PAO/MEL or the oxidative formation of a disulfide bridge by H2O2 involving the same Cys residues causes the inactivation. Our data implicate a possible involvement of thioredoxin in the redox control of CaN activity under physiological conditions. The low temperature EPR spectrum of the native enzyme was consistent with a Fe3+,Zn2+ dinuclear centre. Upon H2O2 -mediated inactivation of the enzyme no significant changes in the EPR spectrum were observed ruling out that Fe2+ is present in the active enzyme and that the dinuclear metal centre is the target for the oxidative inactivation of CaN. [source]


    Cloning and sequence analysis of cnaA gene encoding the catalytic subunit of calcineurin from Aspergillus oryzae

    FEMS MICROBIOLOGY LETTERS, Issue 1 2001
    Praveen Rao Juvvadi
    Abstract Calcineurin has been implicated in ion-homeostasis, stress adaptation in yeast and for hyphal growth in filamentous fungi. Genomic DNA and cDNA encoding the catalytic subunit of calcineurin (cnaA) were isolated from Aspergillus oryzae. The cnaA open reading frame extended to 1727 bp and encoded a putative protein of 514 amino acids. Comparative analysis of the nucleotide sequence of cnaA genomic DNA and cDNA confirmed the presence of three introns and a highly conserved calmodulin binding domain. The deduced amino acid sequence was homologous to calcineurin A from Aspergillus nidulans (92%), Neurospora crassa (84%), human (67%), Saccharomyces cerevisiae (58%) and Schizosaccharomyces pombe (54%). Further, A. oryzae cnaA cDNA complemented S. cerevisiae calcineurin disruptant strain (,cmp1,cmp2), which was not viable in the presence of high concentrations of NaCl (1.2 M) and at alkaline pH 8.5. [source]


    Calcineurin is implicated in the regulation of the septation initiation network in fission yeast

    GENES TO CELLS, Issue 10 2002
    Yabin Lu
    Background: In fission yeast, calcineurin has been implicated in cytokinesis because calcineurin-deleted cells form multiple septa and cell separation is impeded. However, this mechanism remains unclear. Results: We screened for mutations that confer syn-thetic lethality with calcineurin deletion and isolated a mutant, its10-1/cdc7-i10, a novel allele of the cdc7+ gene involved in the septation initiation network (SIN). The mutation created a termination codon, resulting in the truncation of Cdc7 by 162 amino acids, which is not localized in the spindle pole body. Following treatment with the immune suppressive drug FK506, cdc7-i10 and the original cdc7-24 mutant cells showed highly elongated multinuclear morphology with few visible septa, closely resembling the phenotype at the restrictive temperature. Other SIN mutants, cdc11, spg1, sid2 and mob1 showed similar phenotypes following FK506 treatment. Consistent with this, expression of the constitutively active calcineurin suppressed the growth defects and septum initiation deficiency of these SIN mutants at the restrictive temperature. Moreover, electron microscopy revealed that calcineurin-deleted cells had very thick multiple septa which were partially and ectopically formed. Conclusion: These results suggest that calcineurin is involved in the regulation of the SIN pathway, and is required for the proper formation and maturation of the septum in fission yeast. [source]


    Calcineurin phosphatase in signal transduction: lessons from fission yeast

    GENES TO CELLS, Issue 7 2002
    Reiko Sugiura
    Calcineurin (protein phosphatase 2B), the only serine/threonine phosphatase under the control of Ca2+/calmodulin, is an important mediator in signal transmission, connecting the Ca2+ -dependent signalling to a wide variety of cellular responses. Furthermore, calcineurin is specifically inhibited by the immunosuppressant drugs cyclosporin A and tacrolimus (FK506), and these drugs have been a powerful tool for identifying many of the roles of calcineurin. Calcineurin is enriched in the neural tissues, and also distributes broadly in other tissues. The structure of the protein is highly conserved from yeast to man. The combined use of powerful genetics and of specific calcineurin inhibitors in fission yeast Schizosaccharomyces pombe (S. pombe) identified new components of the calcineurin pathway, and defined new roles of calcineurin in the regulation of the many cellular processes. Recent data has revealed functional interactions in which calcineurin phosphatase is involved, such as the cross-talk between the Pmk1 MAP kinase signalling, or the PI signalling. Calcineurin also participates in membrane traffic and cytokinesis of fission yeast through its functional connection with members of the small GTPase Rab/Ypt family, and Type II myosin, respectively. These findings highlight the potential of fission yeast genetic studies to elucidate conserved elements of signal transduction cascades. [source]


    Calcineurin Inhibition Ameliorates Structural, Contractile, and Electrophysiologic Consequences of Postinfarction Remodeling

    JOURNAL OF CARDIOVASCULAR ELECTROPHYSIOLOGY, Issue 9 2001
    LILI DENG M.S.
    Calcineurin Inhibition and Postinfarction Remodeling.Introduction: After myocardial infarction (MI), the heart undergoes an adaptive remodeling process characterized by hypertrophy of the noninfarcted myocardium. Calcineurin, a Ca2+, calmodulin-regulated phosphatase, has been shown to participate in hypertrophic signal transduction. Methods and Results: We investigated the effects of calcineurin inhibition by cyclosporin A on key structural, contractile, and electrophysiologic alterations of post-MI remodeling. Male Sprague-Dawley rats were divided into four groups: (1) sham-operated; (2) sham + cyclosporin A; (3) post-MI (left anterior descending coronary artery ligation); and (4) MI + cyclosporin A. Cyclosporin A (25 mg/kg/day) was initiated 2 days before surgery and continued for 30 days. Hypertrophy was evaluated by echocardiography and by changes in membrane capacitance of isolated myocytes from noninfarcted left ventricle (LV). The effects of cyclosporin A on hemodynamics and cardiac dimensions were investigated, and changes in diastolic function were correlated with changes in protein phosphatase 1 activity and the basal level of phosphorylated phospholamban. The effects of cyclosporin A on Kv4.2/Kv4.3 genes expression and transient outward K + current (Ito) density also were evaluated. One of 12 rats in the post-MI group and 2 of 12 rats in the post-MI + cyclosporin A group died within 48 hours after MI. There were no late deaths in either MI group. There was no evidence of heart failure (lung congestion and/or pleural effusion) in the two groups 4 weeks post-MI. Calcineurin phosphatase activity increased 1.9-fold in post-MI remodeled LV myocardium, and cyclosporin A administration resulted in an 86% decrease in activity. There were statistically significant decreases of LV end-diastolic pressure, LV end-diastolic diameter, and LV relative wall thickness in the post-MI + cyclosporin A group compared with the post-MI group. On the other hand, there was no significant difference in LV end-systolic diameter or peak rate of LV pressure increase between the two post-MI groups. Protein phosphatase 1 activity was elevated by 36% in the post-MI group compared with sham, and this correlated with a 79% decrease in basal level of p16, phospholamban. In the post-MI + cyclosporin A group, the increase in protein phosphatase 1 activity was much less (18% vs 36%; P < 0.05), and the decrease in basal level of p16-phospholamban was markedly ameliorated (20% vs 79%; P < 0.01). The decreases in mRNA levels of Kv4.2 and Kv4.3 and Ito density in the LV of the post-MI + cyclosporin A group were significantly less compared with the post-MI group. Conclusion: Our results show that calcineurin inhibition by cyclosporin A partially ameliorated post-MI remodeled hypertrophy, diastolic dysfunction, decrease in basal level of phosphorylated phospholamban, down-regulation of key K + genes expression, and decrease of K + current, with no adverse effects on systolic function or mortality in the first 4 weeks after MI. [source]


    Calcineurin regulatory subunit is essential for virulence and mediates interactions with FKBP12,FK506 in Cryptococcus neoformans

    MOLECULAR MICROBIOLOGY, Issue 4 2001
    Deborah S. Fox
    Calcineurin is a Ca2+,calmodulin-regulated protein phosphatase that is the target of the immunosuppressive drugs cyclosporin A and FK506. Calcineurin is a heterodimer composed of a catalytic A and a regulatory B subunit. In previous studies, the calcineurin A homologue was identified and shown to be required for growth at 37°C and hence for virulence of the pathogenic fungus Cryptococcus neoformans. Here, we identify the gene encoding the calcineurin B regulatory subunit and demonstrate that calcineurin B is also required for growth at elevated temperature and virulence. We show that the FKR1-1 mutation, which confers dominant FK506 resistance, results from a 6 bp duplication generating a two-amino-acid insertion in the latch region of calcineurin B. This mutation was found to reduce FKBP12,FK506 binding to calcineurin both in vivo and in vitro. Molecular modelling based on the FKBP12,FK506,calcineurin crystal structure illustrates how this mutation perturbs drug interactions with the phosphatase target. In summary, our studies reveal a central role for calcineurin B in virulence and antifungal drug action in the human fungal pathogen C. neoformans. [source]


    ORIGINAL ARTICLE: Calcineurin/NFAT Pathway: A Novel Regulator of Parturition

    AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 1 2009
    Chisa Tabata
    Problem, The oxytocin (OT),oxytocin receptor (OTR) system plays an important role in mammalian parturition. However, we found OTR-deficient (OTRKO) mice are fertile and deliver at term without birth defects, thus alternative pathways inducing parturition can be hypothesized. Methods of study, We tested the gene expression profile of OTRKO mice using suppressive subtractive hybridization, and focused on the calcineurin/nuclear factor of activated T cells (NFAT) pathway. We examined the expression and localization of this pathway in mouse parturition. Results, Calcineurin and NFATc1 were detected in the decidua of pregnant uteri at term using immunohistochemistry (IHC). We identified higher activation levels of NFATc1 in wild type (WT) than in OTRKO mice and increased calcineurin A and NFATc1 mRNA levels during pregnancy. Moreover, injection of FK506, the inhibitor of this pathway, prolonged the delivery of the first pup. Conclusion, Our findings suggested that the calcineurin/NFAT pathway might play a substantial role in initiation of labor. [source]


    Outcomes Following De Novo CNI-Free Immunosuppression After Heart Transplantation: A Single-Center Experience

    AMERICAN JOURNAL OF TRANSPLANTATION, Issue 1 2009
    A. S. Leet
    Renal impairment at the time of heart transplantation complicates the choice of subsequent immunosuppressive therapy. Calcineurin (CNI)-free regimens utilizing proliferation signal inhibitors (PSI) may mitigate against nephrotoxicity in this group; however, their effectiveness remains unclear. We present our 7-year experience with de novo CNI-free, PSI-based immunosuppression after heart transplantation. Of the 152 patients transplanted between July 1999 and July 2006, de novo immunosuppression regimens were 49 CNI-free, PSI-based, 88 CNI, 15 combination of CNI+PSI. Pretransplant creatinine clearance improved within 6 months in the PSI group (0.69 ± 0.34 mL/s vs. 1.00 ± 0.54 mL/s, p < 0.05) but not the CNI (1.32 ± 0.54 mL/s vs. 1.36 ± 0.53 mL/s, p = ns) or CNI+PSI (1.20 ± 0.24 mL/s vs. 1.20 ± 0.41 mL/s, p = ns) groups. The PSI group had more episodes of early (,6 months) acute rejection, bacterial or fungal infections and pleural effusions but less CMV infection (p < 0.05 for all comparisons). Early CNI addition occurred in 37% of the PSI group for acute rejection. 33% of the entire cohort changed immunosuppression regimens over 3.6 ± 2.2 years follow-up. De novo CNI-free, PSI-based immunosuppression in patients with significant renal dysfunction allowed significant posttransplantation renal recovery but with increased early acute rejection, bacterial and fungal infections and pleural effusions. [source]


    Immunopotentiation on murine spleen lymphocytes induced by polysaccharide fraction of Panax ginseng via upregulating calcineurin activity

    APMIS, Issue 4 2010
    SONG-DONG ZHANG
    Zhang S-D, Yin Y-X, Wei Q. Immunopotentiation on murine spleen lymphocytes induced by polysaccharide fraction of Panax ginseng via upregulating calcineurin activity. APMIS 2010; 118: 288,96. Calcineurin (CN), a unique Ca2+/calmodulin (CaM)-dependent serine/threonine protein phosphatase, plays a pivotal role in the activation and proliferation of T lymphocytes. Based on the effective molecular screening model established in our laboratory, we found that a part of polysaccharides from the stem and leaves of Panax ginseng, termed PGP-SL, could activate CN activity. Subsequently, we investigated whether PGP-SL also has immunological competence on murine spleen lymphocytes. In the present study, we demonstrated that PGP-SL could significantly promote in vitro spleen lymphocyte proliferation in the absence of either concanavalin A or LPS in a concentration-dependent manner at concentrations ranging from 100 to 500 ,g/ml (p < 0.001). In addition, the proliferation of cyclosporin A (CsA)-treated spleen lymphocytes was also significantly promoted in the same pattern (p < 0.001); the production of IL-2 was elevated and the effect appeared as early as 24 h after PGP-SL treatment. The results of RT-PCR also indicated that the IL-2 mRNA level was markedly enhanced, particularly at PGP-SL concentrations of 300 and 500 ,g/ml, and Fura-2/AM fluorescence probe analysis showed that PGP-SL could dramatically increase the intracellular free calcium concentration of spleen lymphocytes, i.e. [Ca2+]i was significantly increased by approximately 181 and 107% at 300 and 500 ,g/ml of PGP-SL, respectively. However, this effect could be totally inhibited by verapamil treatment. Taking our results together, we suggest that PGP-SL exhibits immunopotentiation effects on murine spleen lymphocytes by the Ca2+,CN,NFAT,IL-2 signaling pathway. [source]


    Intracellular sodium modulates the state of protein kinase C phosphorylation of rat proximal tubule Na+,K+ -ATPase

    ACTA PHYSIOLOGICA, Issue 2 2002
    F. R. IBARRA
    ABSTRACT The natriuretic hormone dopamine and the antinatriuretic hormone noradrenaline, acting on , -adrenergic receptors, have been shown to bidirectionally modulate the activity of renal tubular Na+,K+ -adenosine triphosphate (ATPase). Here we have examined whether intracellular sodium concentration influences the effects of these bidirectional forces on the state of phosphorylation of Na+,K+ -ATPase. Proximal tubules dissected from rat kidney were incubated with dopamine or the , -adrenergic agonist, oxymetazoline, and transiently permeabilized in a medium where sodium concentration ranged between 5 and 70 mM. The variations of sodium concentration in the medium had a proportional effect on intracellular sodium. Dopamine and protein kinase C (PKC) phosphorylate the catalytic subunit of rat Na+,K+ -ATPase on the Ser23 residue. The level of PKC induced Na+,K+ -ATPase phosphorylation was determined using an antibody that only recognizes Na+,K+ -ATPase, which is not phosphorylated on its PKC site. Under basal conditions Na+,K+ -ATPase was predominantly in its phosphorylated state. When intracellular sodium was increased, Na+,K+ -ATPase was predominantly in its dephosphorylated state. Phosphorylation of Na+,K+ -ATPase by dopamine was most pronounced when intracellular sodium was high, and dephosphorylation by oxymetazoline was most pronounced when intracellular sodium was low. The oxymetazoline effect was mimicked by the calcium ionophore A23187. An inhibitor of the calcium-dependent protein phosphatase, calcineurin, increased the state of Na+,K+ -ATPase phosphorylation. The results imply that phosphorylation of renal Na+,K+ -ATPase activity is modulated by the level of intracellular sodium and that this effect involves PKC and calcium signalling pathways. The findings may have implication for the regulation of salt excretion and sodium homeostasis. [source]


    Insulin release and suppression by tacrolimus, rapamycin and cyclosporin A are through regulation of the ATP-sensitive potassium channel

    DIABETES OBESITY & METABOLISM, Issue 6 2001
    D. K. Fuhrer
    Summary Aim By focusing on the pancreatic , cell response to tacrolimus, cyclosporin A (CsA) and rapamycin we hoped to identify immunophilin, calcineurin and/or novel mechanism involvement and advance the understanding of immunosuppressant regulated insulin control. Methods A glucose responsive , cell model was established in which the glucose response was blocked by immunosuppressant treatment and this model was used to further characterise this effect. Quantification of insulin release to immunosuppressants and specific inhibitors was used to identify the mechanism involved. Results It was found that upon the addition of tacrolimus, rapamycin, or CsA, rapid and significant exocytosis of cellular insulin was seen. A dose response study of this effect revealed optimal concentration windows of 50, 80 nm for tacrolimus, 100,300 nm for rapamycin, and 7,12 mm for CsA in RIN-5F cells. Optimal insulin release for HIT-T15 cells was similar. Additional experiments demonstrate that immunosuppressant pretreatment blocked the subsequent immunosuppressant induced insulin release but not that of a thapsigargin control, suggesting that suppression and release are non-toxic, specific and in the same pathway. Further experiments showed that this insulin release was a calcium dependent process, which was blocked by inhibitors of l -type calcium channels. Continued studies showed that the specific ATP-sensitive potassium channel agonist diazoxide (150 mm) also blocked immunosuppressant-induced insulin release. Conclusions A model that fits this data is a novel calcineurin-independent immunophilin mediated partial closing of the ATP-sensitive potassium channel, which would lead to an initial insulin release but would reduce subsequent responses through this pathway. [source]


    NF-ATc2 induces apoptosis in Burkitt's lymphoma cells through signaling via the B cell antigen receptor

    EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 1 2003
    Eisaku Kondo
    Abstract Cross-linking of the B cell antigen receptor (BCR) with an anti-IgM antibody has been shown to induce dramatic apoptosis in type I Burkitt's lymphoma (BL) cells. However, the apoptotic mechanism triggered via BCR remains unknown. Here we reports a mechanism of BCR ligation-induced apoptosis involving protein phosphatase calcineurin and its specific substrate, transcriptional factor NF-AT. In response to BCR cross-linking, endogenous calcineurin was rapidly activated, and this facilitated nuclear translocation of NF-ATc2, a subtype of NF-AT members. Interestingly, nuclear-imported NF-ATc2 functioned pro-apoptotically in BL cells. The effect of NF-ATc2 was efficiently blocked with FK506, which prevented its nuclear translocation through inactivation of calcineurin. In addtion, TR3 induction during BCR cross-linking was reduced by FK506 and the VIVIT peptide, which is a highly selective inhibitor for NF-AT. This strongly suggests that activation of NF-ATc2 by calcineurin is essential for TR3 recruitment, and that TR3 can be considered as a candidate for death effector in BCR-mediated apoptosis. Therefore, NF-ATc2 plays a crucial role in BCR-mediated apoptosis in type IBL, providing greater insight into unique BL characteristics through BCR signaling. [source]


    Bidirectional synaptic plasticity as a consequence of interdependent Ca2+ -controlled phosphorylation and dephosphorylation pathways

    EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2003
    Pablo D'Alcantara
    Abstract Postsynaptic Ca2+ signals of different amplitudes and durations are able to induce either long-lasting potentiation (LPT) or depression (LTD). The bidirectional character of synaptic plasticity may result at least in part from an increased or decreased responsiveness of the glutamatergic ,-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPA-R) due to the modification of conductance and/or channel number, and controlled by the balance between the activities of phosphorylation and dephosphorylation pathways. AMPA-R depression can be induced by a long-lived Ca2+ signal of moderate amplitude favouring the activation of the dephosphorylation pathway, whereas a shorter but higher Ca2+ signal would induce AMPA-R potentiation resulting from the preferential activation of the phosphorylation pathway. Within the framework of a model involving calcium/calmodulin-dependent protein kinase II (CaMKII), calcineurin (PP2B) and type 1 protein phosphatase (PP1), we aimed at delineating the conditions allowing a biphasic U-shaped relationship between AMPA-R and Ca2+ signal amplitude, and thus bidirectional plasticity. Our theoretical analysis shows that such a property may be observed if the phosphorylation pathway: (i) displays higher cooperativity in its Ca2+ -dependence than the dephosphorylation pathway; (ii) displays a basal Ca2+ -independent activity; or (iii) is directly inhibited by the dephosphorylation pathway. Because the experimentally observed inactivation of CaMKII by PP1 accounts for this latter characteristic, we aimed at verifying whether a realistic model using reported parameters values can simulate the induction of either LTP or LTD, depending on the time and amplitude characteristics of the Ca2+ signal. Our simulations demonstrate that the experimentally observed bidirectional nature of Ca2+ -dependent synaptic plasticity could be the consequence of the PP1-mediated inactivation of CaMKII. [source]


    A modulatory role for protein phosphatase 2B (calcineurin) in the regulation of Ca2+ entry

    EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2000
    J. Russell Burley
    Abstract The Ca2+/calmodulin-dependent protein phosphatase 2B (PP2B) also known as calcineurin (CN) has been implicated in the Ca2+ -dependent inactivation of Ca2+ channels in several cell types. To study the role of calcineurin in the regulation of Ca2+ -channel activity, phosphatase expression was altered in NG108-15 cells by transfection of sense and antisense plasmid constructs carrying the catalytic subunit of human PP2B,3. Relative to mock-transfected (wild-type) controls, cells overexpressing calcineurin showed dramatically reduced high-voltage-activated Ca2+ currents which were recoverable by the inclusion of 1 ,m FK506 in the patch pipette. Conversely, in cells with reduced calcineurin expression, high-voltage-activated Ca2+ currents were larger relative to controls. Additionally in these cells, low-voltage-activated currents were significantly reduced. Analysis of high-voltage-activated Ca2+ currents revealed that the kinetics of inactivation were significantly accelerated in cells overexpressing calcineurin. Following the delivery of a train of depolarizing pulses in experiments designed to produce large-scale Ca2+ influx across the cell membrane, Ca2+ -dependent inactivation of high-voltage-activated Ca2+ currents was increased in sense cells, and this increase could be reduced by intracellular application of 1 m m BAPTA or 1 ,m FK506. These data support a role of calcineurin in the negative feedback regulation of Ca2+ entry through voltage-operated Ca2+ channels. [source]


    N-methyl- d -aspartate enhancement of the glycine response in the rat sacral dorsal commissural neurons

    EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 5 2000
    Tian-.
    Abstract The effect of N-methyl- d -aspartate (NMDA) on the glycine (Gly) response was examined in neurons acutely dissociated from the rat sacral dorsal commissural nucleus (SDCN) using the nystatin-perforated patch-recording configuration under voltage-clamp conditions. The application of 100 ,m NMDA to SDCN neurons reversibly potentiated Gly-activated Cl, currents (IGly) without affecting the Gly binding affinity and the reversal potential of IGly. A selective NMDA receptor antagonist, APV (100 ,m), blocked the NMDA-induced potentiation of IGly, whereas 50 ,m CNQX, a non-NMDA receptor antagonist, did not. The potentiation effect was reduced when NMDA was applied in a Ca2+ -free extracellular solution or in the presence of BAPTA AM, and was independent of the activation of voltage-dependent Ca2+ channels. Pretreatment with KN-62, a selective Ca2+,calmodulin-dependent protein kinase II (CaMKII) inhibitor, abolished the NMDA action. Inhibition of calcineurin (CaN) further enhanced the NMDA-induced potentiation of IGly. In addition, the GABAA receptor-mediated currents were suppressed by NMDA receptor activation in the SDCN neurons. The present results show that Ca2+ entry through NMDA receptors modulates the Gly receptor function via coactivation of CaMKII and CaN in the rat SDCN neurons. This interaction may represent one of the important regulatory mechanisms of spinal nociception. The results also suggest that GABAA and Gly receptors may be subject to different intracellular modulatory pathways. [source]


    Localization of the A kinase anchoring protein AKAP79 in the human hippocampus

    EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 4 2000
    Attila Sík
    Abstract The phosphorylation state of the proteins, regulated by phosphatases and kinases, plays an important role in signal transduction and long-term changes in neuronal excitability. In neurons, cAMP-dependent protein kinase (PKA), protein kinase C (PKC) and calcineurin (CN) are attached to a scaffold protein, A kinase anchoring protein (AKAP), thought to anchor these three enzymes to specific sites of action. However, the localization of AKAP, and the predicted sites of linked phosphatase and kinase activities, are still unknown at the fine structural level. In the present study, we investigated the distribution of AKAP79 in the hippocampus from postmortem human brains and lobectomy samples from patients with intractable epilepsy, using preembedding immunoperoxidase and immunogold histochemical methods. AKAP79 was found in the CA1, presubicular and subicular regions, mostly in pyramidal cell dendrites, whereas pyramidal cells in the CA3, CA2 regions and dentate granule cells were negative both in postmortem and in surgical samples. In some epileptic cases, the dentate molecular layer and hilar interneurons also became immunoreactive. At the subcellular level, AKAP79 immunoreactivity was present in postsynaptic profiles near, but not attached to, the postsynaptic density of asymmetrical (presumed excitatory) synapses. We conclude that the spatial selectivity for the action of certain kinases and phosphatases regulating various ligand- and voltage-gated channels may be ensured by the selective presence of their anchoring protein, AKAP79, at the majority of glutamatergic synapses in the CA1, but not in the CA2/CA3 regions, suggesting profound differences in signal transduction and long-term synaptic plasticity between these regions of the human hippocampus. [source]


    Parvalbumin deficiency in fast-twitch muscles leads to increased ,slow-twitch type' mitochondria, but does not affect the expression of fiber specific proteins

    FEBS JOURNAL, Issue 1 2006
    Peter Racay
    Parvalbumin (PV), a small cytosolic protein belonging to the family of EF-hand calcium-binding proteins, is highly expressed in mammalian fast-twitch muscle fibers. By acting as a ,slow-onset' Ca2+ buffer, PV does not affect the rapid contraction phase, but significantly contributes to increase the rate of relaxation, as demonstrated in PV,/, mice. Unexpectedly, PV,/, fast-twitch muscles were considerably more resistant to fatigue than the wild-type fast-twitch muscles. This effect was attributed mainly to the increased fractional volume of mitochondria in PV,/, fast-twitch muscle, extensor digitorum longus, similar to levels observed in the slow-twitch muscle, soleus. Quantitative analysis of selected mitochondrial proteins, mitochondrial DNA-encoded cytochrome oxidase c subunit I and nuclear DNA-encoded cytochrome oxidase c subunit Vb and F1-ATPase subunit , revealed the PV,/,tibialis anterior mitochondria composition to be almost identical to that in wild-type soleus, but not in wild-type fast-twitch muscles. Northern and western blot analyses of the same proteins in different muscle types and in liver are indicative of a complex regulation, probably also at the post-transcriptional level. Besides the function in energy metabolism, mitochondria in both fast- and slow-twitch muscles act as temporary Ca2+ stores and are thus involved in the shaping of Ca2+ transients in these cells. Previously observed altered spatio-temporal aspects of Ca2+ transients in PV,/, muscles are sufficient to up-regulate mitochondria biogenesis through the probable involvement of both calcineurin- and Ca2+/calmodulin-dependent kinase II-dependent pathways. We propose that ,slow-twitch type' mitochondria in PV,/, fast muscles are aimed to functionally replace the slow-onset buffer PV based on similar kinetic properties of Ca2+ removal. [source]


    7-Ketocholesterol-induced apoptosis

    FEBS JOURNAL, Issue 12 2005
    Involvement of several pro-apoptotic but also anti-apoptotic calcium-dependent transduction pathways
    Oxysterols, and particularly 7-ketocholesterol, appear to be strongly involved in the physiopathology of atherosclerosis. These molecules are suspected to be cytotoxic to the cells of the vascular wall and monocytes/macrophages, particularly by inducing apoptosis. Previous studies have demonstrated that 7-ketocholesterol-induced apoptosis is triggered by a sustained increase of cytosolic-free Ca2+, which elicits the mitochondrial pathway of apoptosis by activation of the calcium-dependent phosphatase calcineurin, leading to dephosphorylation of the ,BH3 only' protein BAD. However, thorough study of the results suggests that other pathways are implicated in 7-ketocholesterol-induced cytotoxicity. In this study, we demonstrate the involvement of two other calcium-dependent pathways during 7-ketocholesterol-induced apoptosis. The activation of the MEK,ERK pathway by the calcium-dependent tyrosine kinase PYK 2, a survival pathway which delays apoptosis as shown by the use of the MEK inhibitor U0126, and a pathway involving another pro-apoptotic BH3 only protein, Bim. Indeed, 7-ketocholesterol treatment of human monocytic THP-1 cells induces the release of Bim-LC8 from the microtubule-associated dynein motor complex, and its association with Bcl-2. Therefore, it appears that 7-ketocholesterol-induced apoptosis is a complex phenomenon resulting from calcium-dependent activation of several pro-apoptotic pathways and also one survival pathway. [source]


    Inactivation of calcineurin by hydrogen peroxide and phenylarsine oxide

    FEBS JOURNAL, Issue 5 2000
    Evidence for a dithiol, disulfide equilibrium, implications for redox regulation
    Calcineurin (CaN) is a Ca2+ -and calmodulin (CaM)-dependent serine/threonine phosphatase containing a dinuclear Fe,Zn center in the active site. Recent studies have indicated that CaN is a possible candidate for redox regulation. The inactivation of bovine brain CaN and of the catalytic CaN A-subunit from Dictyostelium by the vicinal dithiol reagents phenylarsine oxide (PAO) and melarsen oxide (MEL) and by H2O2 was investigated. PAO and MEL inhibited CaN with an IC50 of 3,8 µm and the inactivation was reversed by 2,3-dimercapto-1-propane sulfonic acid. The treatment of isolated CaN with hydrogen peroxide resulted in a concentration-dependent inactivation. Analysis of the free thiol content performed on the H2O2 inactivated enzyme demonstrated that only two or three of the 14 Cys residues in CaN are modified. The inactivation of CaN by H2O2 could be reversed with 1,4-dithiothreitol and with the dithiol oxidoreductase thioredoxin. We propose that a bridging of two closely spaced Cys residues in the catalytic CaN A-subunit by PAO/MEL or the oxidative formation of a disulfide bridge by H2O2 involving the same Cys residues causes the inactivation. Our data implicate a possible involvement of thioredoxin in the redox control of CaN activity under physiological conditions. The low temperature EPR spectrum of the native enzyme was consistent with a Fe3+,Zn2+ dinuclear centre. Upon H2O2 -mediated inactivation of the enzyme no significant changes in the EPR spectrum were observed ruling out that Fe2+ is present in the active enzyme and that the dinuclear metal centre is the target for the oxidative inactivation of CaN. [source]


    Cloning and sequence analysis of cnaA gene encoding the catalytic subunit of calcineurin from Aspergillus oryzae

    FEMS MICROBIOLOGY LETTERS, Issue 1 2001
    Praveen Rao Juvvadi
    Abstract Calcineurin has been implicated in ion-homeostasis, stress adaptation in yeast and for hyphal growth in filamentous fungi. Genomic DNA and cDNA encoding the catalytic subunit of calcineurin (cnaA) were isolated from Aspergillus oryzae. The cnaA open reading frame extended to 1727 bp and encoded a putative protein of 514 amino acids. Comparative analysis of the nucleotide sequence of cnaA genomic DNA and cDNA confirmed the presence of three introns and a highly conserved calmodulin binding domain. The deduced amino acid sequence was homologous to calcineurin A from Aspergillus nidulans (92%), Neurospora crassa (84%), human (67%), Saccharomyces cerevisiae (58%) and Schizosaccharomyces pombe (54%). Further, A. oryzae cnaA cDNA complemented S. cerevisiae calcineurin disruptant strain (,cmp1,cmp2), which was not viable in the presence of high concentrations of NaCl (1.2 M) and at alkaline pH 8.5. [source]


    RESEARCH ARTICLE: Fungicidal activity of amiodarone is tightly coupled to calcium influx

    FEMS YEAST RESEARCH, Issue 3 2008
    Sabina Muend
    Abstract The antiarrhythmic drug amiodarone has microbicidal activity against fungi, bacteria and protozoa. In Saccharomyces cerevisiae, amiodarone triggers an immediate burst of cytosolic Ca2+, followed by cell death markers. Ca2+ transients are a common response to many forms of environmental insults and toxic compounds, including osmotic and pH shock, endoplasmic reticulum stress, and high levels of mating pheromone. Downstream signaling events involving calmodulin, calcineurin and the transcription factor Crz1 are critical in mediating cell survival in response to stress. In this study we asked whether amiodarone induced Ca2+ influx was beneficial, toxic or a bystander effect unrelated to the fungicidal effect of the drug. We show that downregulation of Ca2+ channel activity in stationary phase cells correlates with increased resistance to amiodarone. In actively growing cells, extracellular Ca2+ modulated the size and shape of the Ca2+ transient and directly influenced amiodarone toxicity. Paradoxically, protection was achieved both by removal of external Ca2+ or by adding high levels of CaCl2 (10 mM) to block the drug induced Ca2+ burst. Our results support a model in which the fungicidal activity of amiodarone is mediated by Ca2+ stress, and highlight the pathway of Ca2+ mediated cell death as a promising target for antifungal drug development. [source]


    Calcineurin is implicated in the regulation of the septation initiation network in fission yeast

    GENES TO CELLS, Issue 10 2002
    Yabin Lu
    Background: In fission yeast, calcineurin has been implicated in cytokinesis because calcineurin-deleted cells form multiple septa and cell separation is impeded. However, this mechanism remains unclear. Results: We screened for mutations that confer syn-thetic lethality with calcineurin deletion and isolated a mutant, its10-1/cdc7-i10, a novel allele of the cdc7+ gene involved in the septation initiation network (SIN). The mutation created a termination codon, resulting in the truncation of Cdc7 by 162 amino acids, which is not localized in the spindle pole body. Following treatment with the immune suppressive drug FK506, cdc7-i10 and the original cdc7-24 mutant cells showed highly elongated multinuclear morphology with few visible septa, closely resembling the phenotype at the restrictive temperature. Other SIN mutants, cdc11, spg1, sid2 and mob1 showed similar phenotypes following FK506 treatment. Consistent with this, expression of the constitutively active calcineurin suppressed the growth defects and septum initiation deficiency of these SIN mutants at the restrictive temperature. Moreover, electron microscopy revealed that calcineurin-deleted cells had very thick multiple septa which were partially and ectopically formed. Conclusion: These results suggest that calcineurin is involved in the regulation of the SIN pathway, and is required for the proper formation and maturation of the septum in fission yeast. [source]


    Calcineurin phosphatase in signal transduction: lessons from fission yeast

    GENES TO CELLS, Issue 7 2002
    Reiko Sugiura
    Calcineurin (protein phosphatase 2B), the only serine/threonine phosphatase under the control of Ca2+/calmodulin, is an important mediator in signal transmission, connecting the Ca2+ -dependent signalling to a wide variety of cellular responses. Furthermore, calcineurin is specifically inhibited by the immunosuppressant drugs cyclosporin A and tacrolimus (FK506), and these drugs have been a powerful tool for identifying many of the roles of calcineurin. Calcineurin is enriched in the neural tissues, and also distributes broadly in other tissues. The structure of the protein is highly conserved from yeast to man. The combined use of powerful genetics and of specific calcineurin inhibitors in fission yeast Schizosaccharomyces pombe (S. pombe) identified new components of the calcineurin pathway, and defined new roles of calcineurin in the regulation of the many cellular processes. Recent data has revealed functional interactions in which calcineurin phosphatase is involved, such as the cross-talk between the Pmk1 MAP kinase signalling, or the PI signalling. Calcineurin also participates in membrane traffic and cytokinesis of fission yeast through its functional connection with members of the small GTPase Rab/Ypt family, and Type II myosin, respectively. These findings highlight the potential of fission yeast genetic studies to elucidate conserved elements of signal transduction cascades. [source]


    Calcium/calcineurin signaling in primary cortical astrocyte cultures: Rcan1-4 and cyclooxygenase-2 as NFAT target genes

    GLIA, Issue 7 2008
    Andrea Canellada
    Abstract The calcineurin/nuclear factor of activated T cells (NFAT) signaling pathway mediates important cell responses to calcium, but its activity and function in astrocytes have remained unclear. We show that primary cortical astrocyte cultures express the regulatory and catalytic subunits of the phosphatase calcineurin as well as the calcium-regulated NFAT family members (NFATc1, c2, c3, and c4). NFATs are activated by calcium-mobilizing agents in astrocytes, and this activation is blocked by the calcineurin inhibitor cyclosporine A. Microarray screening identified cyclooxygenase-2 (Cox-2), which is implicated in brain injury, and Rcan 1-4, an endogenous calcineurin inhibitor, as genes up-regulated by calcineurin-dependent calcium signals in astrocytes. Mobilization of intracellular calcium with ionophore potently augments the promoter activity and mRNA and protein expression of Rcan 1-4 and Cox-2 induced by combined treatment with phorbol esters. Moreover, Rcan 1-4 expression is efficiently induced by calcium mobilization alone. For both the genes, the calcium signal component is dependent on calcineurin and is replicated by exogenous expression of a constitutively active NFAT, strongly suggesting that the calcium-induced gene activation is mediated by NFATs. Finally, we report that calcineurin-dependent expression of Cox-2 and Rcan 1-4 is induced by physiological calcium mobilizing agents, such as thrombin, agonists of purinergic and glutamate receptors, and L-type voltage-gated calcium channels. These findings provide insights into calcium-initiated gene transcription in astrocytes, and have implications for the regulation of calcium responses in astrocytes. © 2008 Wiley-Liss, Inc. [source]


    Severe aphthous stomatitis associated with oral calcineurin and mTOR inhibitors

    INTERNATIONAL JOURNAL OF DERMATOLOGY, Issue 1 2010
    Nancy Habib BS
    Background, Aphthous stomatitis, a common mucocutaneous disorder, is a well accepted complication of sirolimus therapy. This association has been reported less frequently with tacrolimus. Case, We present an 11-year old male with Budd-Chiari syndrome who experienced profound worsening of chronic aphthous ulcers after immunosuppressive therapy was changed from tacrolimus to sirolimus. Conclusion, Since these drugs are used widely in the pediatric transplantation population, this report serves to heighten awareness of this debilitating phenomenon, and to stress the importance of exercising caution when sirolimus and tacrolimus are administered in combination to pediatric patients. [source]


    The nonconserved N-terminus of protein phosphatase 2B confers its properties to protein phosphatase 1

    IUBMB LIFE, Issue 2 2009
    Xiu-Jie Xie
    Abstract The protein phosphatase 1 catalytic subunit (PP1c) and the protein phosphatase 2B (PP2B or calcineurin) catalytic subunit (CNA) contain nonconserved N-terminal regions followed by conserved phosphatase cores. To examine the role of the N-termini of these two phosphatases, we substituted the residues 1,8 of PP1c with residues 1,42 of CNA, which is designated CNA(1-42)-PP1(9-330). The activities of CNA(1-42)-PP1(9-330) were similar to those of PP2B and different from those of PP1. The chimera was at least fourfold less sensitive to inhibition by okadaic acid, but was stimulated by nickel ions and chlorogenic acid, characteristics of PP2B not of PP1. These observations suggest that the N-terminus of CNA shifts the properties of PP1 toward those of PP2B. Our findings provide evidence that the nonconserved N-terminus of PP2B not only functions as important regulatory domain but also confers itself particular characteristics. This region may be targeted for regulation of PP2B activities in vivo. © 2008 IUBMB IUBMB Life, 61(2): 178,183, 2009 [source]


    Effects of Cyclosporine on Osteoclast Activity: Inhibition of Calcineurin Activity With Minimal Effects on Bone Resorption and Acid Transport Activity,

    JOURNAL OF BONE AND MINERAL RESEARCH, Issue 3 2003
    John P Williams
    Abstract Cyclosporine results in rapid and profound bone loss in transplant patients, an effect ascribed to osteoclasts. Cyclosporine, complexed with the appropriate immunophilin, inhibits calcineurin (the calcium/calmodulin dependent serine/threonine phosphatase) activity. We tested the hypothesis that cyclosporine inhibits calcineurin activity in osteoclasts, resulting in stimulation of osteoclast activity. We compared the effects of cyclosporine A and the calmodulin antagonist, tamoxifen, on bone resorption by avian osteoclasts. Tamoxifen inhibits bone resorption ,60%, whereas cyclosporine A only inhibited bone resorption 12%. One-hour treatment with 100 nM cyclosporine inhibited osteoclast calcineurin activity 70% in whole cell lysates, whereas 10 ,M tamoxifen only inhibited calcineurin activity 25%. We compared the effects of cyclosporine A and tamoxifen on acid transport activity in isolated membrane vesicles and in isolated membrane vesicles obtained from osteoclasts treated with cyclosporine A or tamoxifen under conditions that inhibit calcineurin activity. Direct addition of cyclosporine A in the acid transport assay, or pretreatment of cells with cyclosporine A followed by membrane isolation, had no effect on acid transport activity in membrane vesicles. In contrast, direct addition of tamoxifen to membranes inhibits acid transport activity, an effect that can be prevented by addition of exogenous calmodulin. Furthermore, acid transport activity was also inhibited in membrane vesicles isolated from cells treated with tamoxifen. In conclusion, cyclosporine A inhibits osteoclast calcineurin activity; however, calcineurin inhibition does not correspond to a significant effect on acid transport activity in isolated membrane vesicles or bone resorption by osteoclasts. [source]


    Prostaglandin F2, inhibits adipocyte differentiation via a G,q-Calcium-Calcineurin-Dependent signaling pathway

    JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2007
    Li Liu
    Abstract Prostaglandin F2, (PGF2,) is a potent physiological inhibitor of adipocyte differentiation, however the specific signaling pathways and molecular mechanisms involved in mediating its anti-adipogenic effects are not well understood. In the current study, we now provide evidence that PGF2, inhibits adipocyte differentiation via a signaling pathway that requires heterotrimeric G-protein G,q subunits, the elevation of the intracellular calcium concentration ([Ca2+]i), and the activation of the Ca2+/calmodulin-regulated serine/threonine phosphatase calcineurin. We show that while this pathway acts to inhibit an early step in the adipogenic cascade, it does not interfere with the initial mitotic clonal expansion phase of adipogenesis, nor does it affect either the expression, DNA binding activity or differentiation-induced phosphorylation of the early transcription factor C/EBP,. Instead, we find that PGF2, inhibits adipocyte differentiation via a calcineurin-dependent mechanism that acts to prevent the expression of the critical pro-adipogenic transcription factors PPAR, and C/EBP,. Furthermore, we demonstrate that the inhibitory effects of PGF2, on both the expression of PPAR, and C/EBP, and subsequent adipogenesis can be attenuated by treatment of preadipocytes with the histone deacetylase (HDAC) inhibitor trichostatin A. Taken together, these results indicate that PGF2, inhibits adipocyte differentiation via a G,q-Ca2+ -calcineurin-dependent signaling pathway that acts to block expression of PPAR, and C/EBP, by a mechanism that appears to involves an HDAC-sensitive step. J. Cell. Biochem. 100: 161,173, 2007. © 2006 Wiley-Liss, Inc. [source]


    NFAT expression in human osteoclasts

    JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2005
    Christopher J. Day
    Abstract Nuclear factor of activated T-cells cytoplasmic (NFATc) is a family of transcription factors originally identified in T-cells. The gene family is currently known to have four members (NFATc1 through NFATc4) which have roles both within and outside the immune system. We show that NFATc1 is the major induced NFAT in human osteoclasts, with expression greatly exceeding that of NFATc2 through NFATc4. In macrophage-like cells in culture, NFATc1 through NFATc4 are expressed at similar low levels. NFATc1 is comprised of five mRNA transcript variants known to encode three different protein isoforms. The mRNA encoding isoform C (mRNA variant 3) was the most expressed with 38 copies per nanogram followed by isoform B (mRNA variant 5) with 17 copies per nanogram of total RNA. Isoform A (mRNA variant 1) and mRNA variants 2 and 4 made up less than 1% of the total NFATc1 expressed. NFATc1 is activated by calcineurin after calcium-calmodulin signalling. The induction of NFATc1 in osteoclasts was not altered in the presence of cyclosporin A, an inhibitor of calcineurin, suggesting that NFATc1 does not participate in autoregulatory activation of its own promoter. The NFATc1 variants expressed by human osteoclasts are not those normally expressed by effector T-cells but are similar to those seen in naïve T-cells. © 2005 Wiley-Liss, Inc. [source]


    A preliminary examination of the role of NFAT 3 in human skin, cultured keratocytes and dermal fibroblasts

    JOURNAL OF CUTANEOUS PATHOLOGY, Issue 9 2010
    Wael I. Al-Daraji
    Background: Ciclosporin A (CsA) is widely utilized for the treatment of inflammatory skin diseases such as psoriasis. The therapeutic effects of CsA are thought to be mediated via its immunosuppressive action on infiltrating lymphocytes in skin lesions. CsA and tacrolimus block T cell activation by inhibiting the phosphatase calcineurin and preventing translocation from the cytoplasm to the nucleus of the transcription factor Nuclear Factor of Activated T cells (NFAT). Methods: RT-PCR and Western Analysis were used to investigate the presence of NFAT-3 mRNA and protein in human keratocytes. Tissue culture of human keratocytes and immunostaining of cells on coverslips and confocal microscopy were used to assess the degree of nuclear localisation of NFAT-3 in cultured cells. Keratome biopsies were taken from patients with psoriasis (lesional and non-lesional skin) and normal skin and immunohistochemistry was used to assess the NFAT-3 localisation in these biopsies using a well characterized anti-NFAT-3 antibody. Results: The NFAT-3 mRNA and protein expression was demonstrated using RT-PCR and Western blotting. The expression of NFAT-3 in human keratocytes and response to different agonists provides perhaps a unique opportunity to examine the regulation, subcellular localization and kinetics of translocation of different NFATs in primary cultured human cells. As with NFAT 1, NFAT 2 and recently NFAT 5, differentiation-promoting agents that increase intracellular calcium concentration induced nuclear translocation of NFAT-3 in cultured keratocytes but with different kinetics. Conclusion: These data provide the first evidence of that NFAT-3 is expressed in normal skin, psoriasis and that NFAT-3 functionally active in human keratocytes and that nuclear translocation of NFAT-3 in human skin cells has different kinetics than NFAT 1 suggesting that NFAT-3 may play an important role in regulation of keratocytes proliferation and differentiation at a different stage. Inhibition of this pathway in human epidermal keratocytes many account, in part for the therapeutic effects of CsA and tacrolimus in skin disorders such as psoriasis. Al-Daraji WI. A preliminary examination of the role of NFAT 3 in human skin, cultured keratocytes and dermal fibroblasts. [source]