Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cyclosporine A (CsA) and FK506, important immunosuppressants, have been shown to inhibit the enzymatic equivalent of the Na(+)-K(+) pump (Na(+), K(+)-ATPase) in renal tissue. A similar effect in the heart may contribute to the adverse effects of these agents that include calcification, contractile dysfunction, and altered calcium handling. However, inhibition of the pump has not been demonstrated in cardiac myocytes. We isolated single ventricular myocytes from control rabbits and from rabbits administered CsA or FK506 for 1 week. Na(+)-K(+) pump current (I(p)) was measured using the whole-cell patch-clamp technique. When patch pipettes contained Na(+) in a concentration ([Na](pip)) near physiological intracellular levels mean I(p) of cardiac myocytes from rabbits with serum CsA levels within the therapeutic range was significantly lower than mean I(p) of cardiac myocytes from controls. Treatment had no effect on I(p) measured using a [Na](pip) expected to nearly saturate intracellular binding sites. The CsA-induced inhibition of I(p) was dependent on the K(+) concentration in pipette solutions. Mean I(p) in myocytes from rabbits with serum levels of FK506 within the therapeutic range was similar to mean I(p) in myocytes from controls, whereas FK506 in a dose inducing serum levels severalfold above the therapeutic range caused significant pump inhibition. Using ion-sensitive microelectrodes we showed the intracellular Na(+) activity in papillary muscles isolated from rabbits treated with CsA was significantly higher than in papillary muscles from control rabbits, indicating that CsA causes pump inhibition in intact myocytes with a physiological intracellular milieu.
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PMID:Cyclosporine and FK506 differentially regulate the sarcolemmal Na(+)-K(+) pump. 1130 73

Sodium tolerance in yeast is enhanced by continuous activation of calcineurin, a Ca(2+)/calmodulin-dependent protein phosphatase that is required for modulation of the Na(+) efflux mechanism. We isolated several salt-tolerant mutations with the treatment of ethylmethane sulfonate under high salt stress. One of the mutations was mapped in the PMR1 gene. Pmr1p, the P-type Ca(2+)-ATPase in the Golgi apparatus, regulates a cytosolic Ca(2+) level in various responses. Cytosolic Ca(2+) concentration in the pmr1 mutant is highly maintained, and thus calcineurin is activated continuously. The treatment of FK506, a specific inhibitor of calcineurin, abolishes the salt-tolerant phenotype of the pmr1 mutant. Activated calcineurin induces the expression of PMR2, encoding the P-type Na(+)-ATPase, through the specific transcription factor, Tcn1p/Crz1p. Also, expression of the PMR2::lacZ reporter gene in the pmr1 mutant was higher than that in wild type. We propose that the pmr1 mutation confers salt tolerance through continuous activation of calcineurin and that Pmr1p might act as a major Ca(2+)-ATPase under high salt stress.
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PMID:Mutation in PMR1, a Ca(2+)-ATPase in Golgi, confers salt tolerance in Saccharomyces cerevisiae by inducing expression of PMR2, an Na(+)-ATPase in plasma membrane. 1138 21

The effect of carbachol (Cch) on intracellular calcium concentration ([Ca2+]i) in eel enterocytes was examined using the fluorescent Ca2+ indicator fura-2. Cch caused a biphasic increase in [Ca2+]i, with an initial spike followed by a progressively decreasing level (over 6 min) to the initial, pre-stimulated, level. The effect of Cch was dose-dependent with a 7.5-fold increase in [Ca2+]i over basal level induced by the maximal dose of Cch (100 microM). In Ca2+-free/EGTA buffer the effect of Cch was less pronounced and the [Ca2+]i returned rapidly to basal levels. The increment of [Ca2+]i was dose-dependently attenuated in cells pre-treated with U73122, a specific inhibitor of phospholipase C, suggesting that the Cch-stimulated increment of [Ca2+]i required inositol triphosphate formation. In the presence of extracellular Ca2+, thapsigargin (TG), a specific microsomal Ca2+-ATPase inhibitor, caused a sustained rise in [Ca2+]i whereas in Ca2+-free medium the increase in [Ca2+]i was transient; in both cases, subsequent addition of Cch was without effect. When 2 mM CaCl2 were added to the cells stimulated with TG or with Cch in Ca2+-free medium, a rapid increase in [Ca2+]i was detected, corresponding to the capacitative Ca2+ entry. Thus, both TG and Cch depleted intracellular Ca2+ stores and stimulated influx of extracellular Ca2+ consistent with capacitative Ca2+ entry. K+ depolarization obtained with increasing concentrations of KCl in the extracellular medium induced a dose-related increase in [Ca2+]i which was blocked by 2 microM nifedipine, a non-specific L-type Ca2+ channel blocker. Nifedipine also changed significantly the height of the Ca2+ transient, and the rate of decrement to the pre-stimulated [Ca2+]i level, indicating that Ca2+ entry into enterocytes also occurs through an L-type voltage-dependent calcium channel pathway. We also show that isolated enterocytes stimulated with increasing Cch concentrations (0.1-1000 microM) showed a dose-dependent inhibition of the Na+/K+-ATPase activity. The threshold decrease was at 1 microM Cch; it reached a maximum at 100 microM (50.5% inhibition) and did not decrease further with the use of higher dose. The effect of Cch on Na+/K+-ATPase activity was dependent on both protein kinase C (PKC) and protein phosphatase calcineurin activation since the PKC inhibitor calphostin C abolished Cch effects, while the calcineurin inhibitor FK506 augmented Cch effect. Collectively, these data establish a functional pathway by which Cch can modulate the activity of the Na+/K+-ATPase through a PKC-dependent (calphostin C-sensitive) pathway and a calcineurin-dependent (FK506-sensitive) pathway.
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PMID:Muscarinic acetylcholine receptor activation induces Ca2+ mobilization and Na+/K+-ATPase activity inhibition in eel enterocytes. 1201 Jun 40

Stress of the endoplasmic reticulum (ER), which is associated with many neurodegenerative conditions, can lead to the elimination of affected cells by apoptosis through only partially understood mechanisms. Thapsigargin, which causes ER stress by inhibiting the ER Ca(2+)-ATPase, was found to not only activate the apoptosis effector caspase-3 but also to cause a large and prolonged increase in the activity of glycogen synthase kinase-3beta (GSK3beta). Activation of GSK3beta was obligatory for thapsigargin-induced activation of caspase-3, because inhibition of GSK3beta by expression of dominant-negative GSK3beta or by the GSK3beta inhibitor lithium blocked caspase-3 activation. Thapsigargin treatment activated GSK3beta by inducing dephosphorylation of phospho-Ser-9 of GSK3beta, a phosphorylation that normally maintains GSK3beta inactivated. Caspase-3 activation induced by thapsigargin was blocked by increasing the phosphorylation of Ser-9-GSK3beta with insulin-like growth factor-1 or with the phosphatase inhibitors okadaic acid and calyculin A, but the calcineurin inhibitors FK506 and cyclosporin A were ineffective. Insulin-like growth factor-1, okadaic acid, calyculin A, and lithium also protected cells from two other inducers of ER stress, tunicamycin and brefeldin A. Thus, ER stress activates GSK3beta through dephosphorylation of phospho-Ser-9, a prerequisite for caspase-3 activation, and this process is amenable to pharmacological intervention.
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PMID:Central role of glycogen synthase kinase-3beta in endoplasmic reticulum stress-induced caspase-3 activation. 1222 24

The effect of antisense oligodeoxynucleotides (ODNs) of plasma membrane Ca(2+)-pumping ATPase (PMCA) on rat aortic vascular smooth muscle cells (VSMCs) in primary culture was examined. More than 80% of the PMCA expressed in cultured VSMCs was the PMCA-1B subtype. Exposed to antisense ODNs against PMCA-1, not only the expression of the PMCA protein but also mRNA of PMCA-1B was diminished in a concentration-dependent manner. Extracellular Na(+)-independent (45)Ca(2+) efflux catalyzed via PMCA was inhibited with antisense ODNs. Both the resting and ionomycin- or ATP-stimulated levels of intracellular Ca(2+) were increased by antisense ODNs. Furthermore, prolonged treatment with antisense ODNs caused apoptosis in VSMCs. The occurrence of apoptosis was inhibited by FK506, a potent immunosuppressant. These results demonstrate that the PMCA was specifically inhibited by antisense ODNs and suggest that PMCA plays an important role in regulation of intracellular Ca(2+) concentrations, especially at the resting condition to prevent an occurrence of apoptosis that may be induced through the activation of calcineurin.
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PMID:Antisense-inhibition of plasma membrane Ca2+ pump induces apoptosis in vascular smooth muscle cells. 1241 87

Elevated levels of free fatty acids (FFA) have been implicated in the pathogenesis of neuronal injury and death induced by cerebral ischemia. This study evaluated the effects of immunosuppressants agents, calcineurin inhibitors and blockade of endoplasmic reticulum (ER) calcium channels on free fatty acid formation and efflux in the ischemic/reperfused (I/R) rat brain. Changes in the extracellular levels of arachidonic, docosahexaenoic, linoleic, myristic, oleic and palmitic acids in cerebral cortical superfusates during four-vessel occlusion-elicited global cerebral ischemia were examined using a cortical cup technique. A 20-min period of ischemia elicited large increases in the efflux of all six FFAs, which were sustained during the 40 min of reperfusion. Cyclosporin A (CsA) and trifluoperazine, which reportedly inhibit the I/R elicited opening of a mitochondrial permeability transition (MPT) pore, were very effective in suppressing ischemia/reperfusion evoked release of all six FFAs. FK506, an immunosuppressant which does not directly affect the MPT, but is a calcineurin inhibitor, also suppressed the I/R-evoked efflux of FFAs, but less effectively than CsA. Rapamycin, a derivative of FK506 which does not inhibit calcineurin, did not suppress I/R-evoked FFA efflux. Gossypol, a structurally unrelated inhibitor of calcineurin, was also effective, significantly reducing the efflux of docosahexaenoic, arachidonic and oleic acids. As previous experiments had implicated elevated Ca(2+) levels in the activation of phospholipases with FFA formation, agents affecting endoplasmic reticulum stores were also evaluated. Dantrolene, which blocks the ryanodine receptor (RyR) channel of the ER, significantly inhibited I/R-evoked release of docosahexaenoic, arachidonic, linoleic and oleic acids. Ryanodine, which can either accentuate or block Ca(2+) release, significantly enhanced ischemia/reperfusion-elicited efflux of linoleic acid, with non-significant increases in the efflux of myristic, arachidonic, palmitic and oleic acids. Xestospongin C, an inhibitor of the inositol triphosphate (IP(3)R) channel, failed to affect I/R-evoked FFA efflux. Thapsigargin, an inhibitor of the Ca(2+)-ATPase ER uptake pump, elicited significant elevations in the efflux of myristic, arachidonic and linoleic acids, in the absence of ischemia. Collectively, the data suggest an involvement of both ER and mitochondrial Ca(2+) stores in the chain of events which lead to PLA(2) activation and FFA formation.
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PMID:Effects of immunosuppressants, calcineurin inhibition, and blockade of endoplasmic reticulum calcium channels on free fatty acid efflux from the ischemic/reperfused rat cerebral cortex. 1244 75

The short-time transcriptional response of yeast cells to a mild increase in external pH (7.6) has been investigated using DNA microarrays. A total of 150 genes increased their mRNA level at least twofold within 45 min. Alkalinization resulted in the repression of 232 genes. The response of four upregulated genes, ENA1 (encoding a Na+-ATPase also induced by saline stress) and PHO84, PHO89 and PHO12 (encoding genes upregulated by phosphate starvation), was characterized further. The alkaline response of ENA1 was not affected by mutation of relevant genes involved in osmotic or oxidative signalling, but was decreased in calcineurin and rim101 mutants. Mapping of the ENA1 promoter revealed two pH-responsive regions. The response of the upstream region was fully abolished by the drug FK506 or mutation of CRZ1 (a transcription factor activated by calcium/calcineurin), whereas the response of the downstream region was essentially calcium independent. PHO84 and PHO12 responses were unaffected in crz1 cells, but required the presence of Pho2 and Pho4. In contrast, part of the alkali-induced expression of PHO89 was maintained in pho4 or pho2 cells, but was fully abolished in a crz1 strain or in the presence of FK506. Heterologous promoters carrying the minimal calcineurin-dependent response elements found in ENA1 or FKS2 were able to drive alkaline pH-induced expression. These results demonstrate that the transcriptional response to alkaline pH involves different signalling mechanisms, and that calcium signalling is a relevant component of this response.
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PMID:The transcriptional response to alkaline pH in Saccharomyces cerevisiae: evidence for calcium-mediated signalling. 1245 18

In this study, we investigated whether nucleoplasmic free Ca2+ in aortic vascular smooth muscle cells (VSMCs) might be independently regulated from cytosolic free Ca2+. Understanding mechanisms and pathways responsible for this regulation is especially relevant given the role of a numerous intranuclear Ca2+-sensitive proteins in transcriptional regulation, apoptosis and cell division. The question of an independent regulatory mechanism remains largely unsettled because the previous use of intensitometric fluorophores (e.g., Fluo-3) has been criticized on technical grounds. To circumvent the potential problem of fluorescence artifact, we utilized confocal laser scanning microscopy to image intracellular Ca2+ movements with the ratiometric fluorophore Indo-1. In cultured rabbit VSMCs, we found sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA) pumps and ryanodine receptor (RyR) Ca2+ channel proteins to be discretely arranged within a perinuclear locus, as determined by fluorescent staining patterns of BODIPY FL thapsigargin and BODIPY FL-X Ry. When intracellular Ca2+ stores were mobilized by addition of thapsigargin (5 microM) and activatory concentrations of ryanodine (1 microM), Indo-1 ratiometric signals were largely restricted to the nucleoplasm. Cytosolic signals, by comparison, were relatively small and even then its spatial distribution was largely perinuclear rather homogeneous. These observations indicate perinuclear RyR and SERCA proteins are intimately involved in regulating VSMC nucleoplasmic Ca2+ concentrations. We also observed a similar pattern of largely nucleoplasmic Ca2+ mobilization upon exposure of cells to the immunosuppressant drug FK506 (tacrolimus), which binds to the RyR-associated immunophillin-binding proteins FKBP12 and FKBP12.6. However, initial FK506-induced nucleoplasmic Ca2+ mobilization was followed by marked reduction of Indo-1 signal intensity close to pretreatment levels. This suggested FK506 exerts both activatory and inhibitory effects upon RyR channels. The latter was reinforced by observed effects of FK506 to only reduce nucleoplasmic Indo-1 signal intensity when added following pretreatment with both activatory and inhibitory concentrations of ryanodine. These latter observations raise the possibility that VSMC nuclei represent an important sink of intracellular Ca2+ and may help explain vasodilatory actions of FK506 observed by others.
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PMID:Nucleoplasmic calcium regulation in rabbit aortic vascular smooth muscle cells. 1273 28

FK506, a calcineurin inhibitor, shows potent neuroprotective effects in animal models such as those of stroke and neurodegenerative diseases. However, the mechanism underlying these neuroprotective effects is unclear. In this study, an in vitro model, in which FK506 protected the cells against cell death, was established and analyzed in detail by pharmacological experiments. Thapsigargin (TG), an inhibitor of endoplasmic reticulum calcium-ATPase, induced SH-SY5Y cell death. FK506 concentration-dependently protected the cells from this type of death. In contrast, FK506 did not suppress SH-SY5Y cell death caused by the following molecules: tunicamycin (TM), an inhibitor of N-linked glycosylation; etoposide (Eto), a topoisomerase II inhibitor; and staurosporine (STS), a phospholipid/calcium-dependent protein kinase inhibitor. Additionally, FK506 did not inhibit TG-induced cell death in either SK-N-MC or HeLa cell lines. FK506 completely inhibited caspase-3 activation and apoptosis caused by TG in a concentration-dependent manner, but not that caused by TM, Eto, and STS. TG did not activate caspase-3 in SK-N-MC cells, although it slightly activated caspase-3 in HeLa cells. FK506 did not change caspase-3 activity in either SK-N-MC or HeLa cell lines. Cyclosporin A, another calcineurin inhibitor, showed the same results as FK506 in this study, whereas rapamycin, an immunosuppressant not associated with calcineurin activity, did not have any effect in this context. Thus, the suppressive effects of FK506 on cell death are specific to SH-SY5Y cells treated with TG and are caused by the inhibition of calcineurin and subsequent suppression of caspase-3 activation. Therefore, an in vitro system using SH-SY5Y cells treated with TG could provide a model reflective of certain aspects of the neuroprotective activity of FK506.
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PMID:Detailed in vitro pharmacological analysis of FK506-induced neuroprotection. 1287 56

Saccharomyces cerevisiae strains lacking the Ppz1 protein phosphatase are salt tolerant and display increased expression of the ENA1 Na(+)-ATPase gene, a major determinant for sodium extrusion, while cells devoid of the similar Ppz2 protein do not show these phenotypes. However, a ppz1 ppz2 mutant displays higher levels of ENA1 expression than the ppz1 strain. We show here that the increased activity of the ENA1 promoter in a ppz1 ppz2 mutant maps to two regions: one region located at -751 to -667, containing a calcineurin-dependent response element (CDRE), and one downstream region (-573 to -490) whose activity responds to intracellular alkalinization. In contrast, the increased ENA1 expression in a ppz1 mutant is mediated solely by an intact calcineurin/Crz1 signaling pathway, on the basis that (i) this effect maps to a single region that contains the CDRE and (ii) it is blocked by the calcineurin inhibitor FK506, as well as by deletion of the CNB1 or CRZ1 gene. The calcineurin dependence of the increased ENA1 expression of a ppz1 mutant would suggest that Ppz1 could negatively regulate calcineurin activity. In agreement with this notion, a ppz1 strain is calcium sensitive, and this mutation does not result in a decrease in the calcium hypertolerance of a cnb1 mutant. It has been shown that ENA1 can be induced by alkalinization of the medium and that a ppz1 ppz2 strain has a higher intracellular pH. However, we present several lines of evidence that show that the gene expression profile of a ppz1 mutant does not involve an alkalinization effect. In conclusion, we have identified a novel role for calcineurin, but not alkalinization, in the control of ENA1 expression in ppz1 mutants.
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PMID:Regulation of ENA1 Na(+)-ATPase gene expression by the Ppz1 protein phosphatase is mediated by the calcineurin pathway. 1455 76


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