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)

The stimulation of human tumor cells overexpressing epidermal growth factor receptor (EGFR) with EGF enhances tumor development and malignancy. Therefore, compounds that modulate the EGF-mediated signal inducing apoptosis in EGFR-overexpressing cells would represent a new class of antitumor drug and might be useful in the treatment of a subset of human tumors. In the course of screening for compounds that induce apoptosis in EGFR-overexpressing human epidermal carcinoma A431 cells from secondary metabolites of microorganisms, we found that vacuolar-type H(+)-ATPase (V-ATPase) inhibitors, such as concanamycin B and destruxin E, induced apoptosis only when the cells were stimulated with EGF. The EGF-dependent apoptosis by V-ATPase inhibitors was not observed in other types of human tumor cells which do not overexpress EGFR. The apoptosis in A431 cells was inhibited by anti-FasL antibody which neutralized the cytotoxic effect of FasL, indicating that the Fas/FasL system was involved. The expression of cell surface FasL was upregulated by stimulation with EGF and increased further by V-ATPase inhibitors. Moreover, EGF inhibited cytotoxic Fas antibody-induced apoptosis, whereas V-ATPase inhibitors disrupted the protective effect of EGF on apoptosis in A431 cells. Taken together, these results suggested that V-ATPase inhibitors induced EGF-dependent apoptosis in A431 cells, possibly through both the enhancement of EGF-induced cell surface expression of FasL and the disruption of an EGF-induced survival signal.
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PMID:Induction of EGF-dependent apoptosis by vacuolar-type H(+)-ATPase inhibitors in A431 cells overexpressing the EGF receptor. 1221 20

Synthetic analogue of the concanamycins, which lacks the hydrogen bond network existing in the concanamycin structure, retains vacuolar-type H(+)-ATPase (V-ATPase) inhibitory activity and induces apoptosis to cancer cells that overexpressing epidermal growth factor receptors (EGFR).
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PMID:Vacuolar-type H(+)-ATPase inhibitory activity of synthetic analogues of the concanamycins: is the hydrogen bond network involving the lactone carbonyl, the hemiacetal hydroxy group, and the C-19 hydroxy group essential for the biological activity of the concanamycins? 1244 68

Secretory activity in blowfly salivary glands is activated by the hormone serotonin. We have investigated the distribution and activity of two cation pumps that are possibly involved with transepithelial ion transport, i.e. Na(+)/K(+)-ATPase and vacuolar-type H(+)-ATPase (V-ATPase). By immunofluorescence labelling of secretory cells, Na(+)/K(+)-ATPase was localized on the basolateral plasma membrane and V-ATPase on the highly folded apical membrane. Activities of both ATPases were probed in salivary gland homogenates by applying specific inhibitors for these ion pumps, namely ouabain and bafilomycin A(1). In control glands, bafilomycin-A(1)-sensitive V-ATPase activity and ouabain-sensitive Na(+)/K(+)-ATPase activity accounted for 36% and 19%, respectively, of the total ATPase activity. V-ATPase activity increased approximately twofold after stimulation with serotonin, whereas Na(+)/K(+)-ATPase activity was not significantly affected. Biochemical assays provided evidence that the serotonin-induced activation of V-ATPase activity was accompanied by a recruitment of peripheral V(1) subunits from the cytosol to the plasma membrane, indicative of the assembly of V(0)V(1) holoenzymes. These data show that a V-ATPase located in the apical plasma membranes of the secretory cells is a component of the apical "potassium pump" that has been identified previously by physiological approaches. The V-ATPase energizes the apical membrane and provides the primary driving force for fuelling a putative K(+)/nH(+) antiporter and, thus, for fluid secretion. Serotonin-induced assembly of V(0)V(1) holoenzymes might constitute a regulatory mechanism for the control of pump activity.
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PMID:Distribution and serotonin-induced activation of vacuolar-type H+-ATPase in the salivary glands of the blowfly Calliphora vicina. 1272 8

The differentiation and functions of osteoclasts (OC) are regulated by osteoblast-derived factors such as receptor activator of NFKB ligand (RANKL) that stimulates OC formation, and a novel secreted member of the TNF receptor superfamily, osteoprotegerin (OPG), that negatively regulates osteoclastogenesis. In examination of the preosteoclast (pOC) culture, pOCs formed without any additives expressed tartrate-resistant acid phosphatase (TRAP), but showed little resorptive activity. pOC treated with RANKL became TRAP-positive OC, which expressed intense vacuolar-type H(+)-ATPase and exhibited prominent resorptive activity. Such effects of RANKL on pOC were completely inhibited by addition of OPG. OPG inhibited ruffled border formation in mature OC and reduced their resorptive activity, and also induced apoptosis of some OC. Although OPG administration significantly reduced trabecular bone loss in the femurs of ovariectomized (OVX) mice, the number of TRAP-positive OC in OPG-administered OVX mice was not significantly decreased. Rather, OPG administration caused the disappearance of ruffled borders and decreased H(+)-ATPase expression in most OC. OPG deficiency causes severe osteoporosis. We also examined RANKL localization and OC induction in periodontal ligament (PDL) during experimental movement of incisors in OPG-deficient mice. Compared to wild-type OPG (+/+) littermates, after force application, TRAP-positive OC were markedly increased in the PDL and alveolar bone was severely destroyed in OPG-deficient mice. In both wild-type and OPG-deficient mice, RANKL expression in osteoblasts and fibroblasts became stronger by force application. These in vitro and in vivo studies suggest that RANKL and OPG are important regulators of not only the terminal differentiation of OC but also their resorptive function. To determine resorptive functions of OC, we further examined the effects of specific inhibitors of H(+)-ATPase, bafilomycin A1, and lysosomal cysteine proteinases (cathepsins), E-64, on the ultrastructure, expression of these enzymes and resorptive functions of cultured OC. In bafilomycin A1-treated cultures, OC lacked ruffled borders, and H(+)-ATPase expression and resorptive activity were significantly diminished. E-64 treatment did not affect the ultrastructure and the expression of enzyme molecules in OC, but significantly reduced resorption lacuna formation, by inhibition of cathepsin activity. Lastly, we examined the expression of H(+)-ATPase, cathepsin K, and matrix metalloproteinase-9 in odontoclasts (OdC) during physiological root resorption in human deciduous teeth, and found that there were no differences in the expression of these molecules between OC and OdC. RANKL was also detected in stromal cells located on resorbing dentine surfaces. This suggests that there is a common mechanism in cellular resorption of mineralized tissues such as bone and teeth.
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PMID:Differentiation and functions of osteoclasts and odontoclasts in mineralized tissue resorption. 1287 16

Secretion in blowfly salivary glands is induced by the neurohormone serotonin and powered by a vacuolar-type H(+)-ATPase (V-ATPase) located in the apical membrane of the secretory cells. We have established a microfluorometric method for analysing pH changes at the luminal surface of the secretory epithelial cells by using the fluorescent dye 5-N-hexadecanoyl-aminofluorescein (HAF). After injection of HAF into the lumen of the tubular salivary gland, the fatty acyl chain of the dye molecule partitions into the outer leaflet of the plasma membrane and its pH-sensitive fluorescent moiety is exposed at the cell surface. Confocal imaging has confirmed that HAF distributes over the entire apical membrane of the secretory cells and remains restricted to this membrane domain. Ratiometric analysis of HAF fluorescence demonstrates that serotonin leads to a reversible dose-dependent acidification at the luminal surface. Inhibition by concanamycin A confirms that the serotonin-induced acidification at the luminal surface is due to H(+) transport across the apical membrane via V-ATPase. Measurements with pH-sensitive microelectrodes corroborate a serotonin-induced luminal acidification and demonstrate that luminal pH decreases by about 0.4 pH units at saturating serotonin concentrations. We conclude that ratiometric measurements of HAF fluorescence provide an elegant method for monitoring V-ATPase-dependent H(+) transport in the blowfly salivary gland in vivo and for analysing the spatiotemporal pattern of pH changes at the luminal surface.
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PMID:Fluorescence measurements of serotonin-induced V-ATPase-dependent pH changes at the luminal surface in salivary glands of the blowfly Calliphora vicina. 1662 52

Certain carboxylic acid groups within the primary structure of proton translocating proteins are thought to be involved in the proton pathway. In this report, the effects of a lipophilic carboxylic acid reactive reagent, N-cyclo-N'(4-dimethylamino-alpha-naphthyl)carbodiimide (NCD-4), on the two types of proton pumps in maize (Zea mays L.) root microsomes were investigated. NCD-4 was found to inhibit the vacuolar-type H(+)-ATPase in microsomal preparations; however, the plasma membrane-type H(+)-ATPase was unaffected. The H(+)-ATPase in highly purified tonoplast vesicles was also inhibited by NCD-4. Inhibition was dependent on the concentration and length of exposure to the reagent. However, there was little, if any, increase in the fluorescence of treated vesicles, indicating few carboxylic acid residues were reacting. Inhibition of the tonoplast H(+)-ATPase by NCD-4 was examined further with a partially purified preparation. The partially purified H(+)-ATPase also showed sensitivity to the NCD-4, supporting the hypothesis that this carboxylic acid reagent is an inhibitor of the tonoplast ATPase from maize roots.
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PMID:N-Cyclo-N'-(4-Dimethylamino-alpha-Naphthyl)Carbodiimide Inhibits Membrane-Bound and Partially Purified Tonoplast ATPase from Maize Roots. 1666 43

Vacuolar-type H(+)-ATPase was solubilized from tonoplasts of mung bean (Vigna radiata L.) and purified on a Mono Q anion-exchange column by fast protein liquid chromatography. The purified enzyme was inactivated by the reactive adenine analog, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). This inactivation was reversed by addition of dithiothreitol (DTT). Inactivation by NBD-Cl was prevented by Mg-ADP, a competitive inhibitor of ATPase. [(14)C]NBD-Cl predominantly modified the 68-kilodalton subunit and the degree of (14)C incorporation was decreased in the presence of Mg-ADP or upon subsequent addition of DTT. The loss of activity followed pseudo first-order kinetics with respect to NBD-Cl concentration, and double log plots of pseudo first-order rate constants versus reagent concentration yielded a straight line with a slope of 0.957. The NBD-modified/inactivated enzyme showed an absorbance maximum at 418 nanometers and a fluorescence emission peak at 515 nanometers. The absorption and fluorescence emission spectra of the NBD-modified enzyme were essentially the same as those of the model compound, N-acetyl-S-NBD cysteine. Absorbance by the modified enzyme at 418 nanometers disappeared upon addition of DTT, which coincided with the restoration of ATPase activity and the decrease in bound [(14)C]NBD-Cl. These findings show that NBD-Cl modifies an essential cysteine residue(s) at or near the catalytic site in the 68-kilodalton subunit of tonoplast H(+)-ATPase and that the modification closely correlates with the loss of ATPase activity.
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PMID:Binding of 7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole to an Essential Cysteine Residue(s) in the Tonoplast H-ATPase from Mung Bean (Vigna radiata L.) Hypocotyls. 1666 36

In this study, the existence and functional activity of a vacuolar-type H(+)-ATPase (vH(+)-ATPase) was explored in primary cultures of sheep ruminal epithelial cells (REC). The mRNA transcripts of the E and B subunits of vH(+)-ATPase were detectable in RNA from REC samples by RT-PCR. Immunoblotting of REC protein extractions with antibodies directed against the B subunit of yeast vH(+)-ATPase revealed a protein band of the expected size (60 kDa). Using the fluorescent indicator BCECF and selective inhibitors (foliomycin, HOE 694, S3226), the contribution of vH(+)-ATPase and Na(+)/H(+) exchanger (NHE) subtype 1 and 3 activity to the regulation of intracellular pH (pH(i)) was determined in nominally HCO(3)(-)-free, HEPES-buffered NaCl medium containing 20 mM of the short-chain fatty acid butyrate as well as after reduction of the extracellular Cl(-) concentration ([Cl(-)](e)) from 136 to 36 mM. The initial pH(i) of REC was 7.4 +/- 0.1 in nominally HCO(3)(-)-free, HEPES-buffered NaCl medium and 7.0 +/- 0.1 after acid loading with butyrate. Selective inhibition of the vH(+)-ATPase with foliomycin decreased pH(i) by 0.19 +/- 0.03 pH units. On the basis of the observed decreases in pH(i) resulting from inhibition of vH(+)-ATPase as well as of subtypes 1 and 3 of NHE, vH(+)-ATPase activity appears to account for approximately 30% of H(+) extrusion, whereas the activities of NHE subtypes 3 and 1 account for 20 and 50% of H(+) extrusion, respectively. Lowering of [Cl(-)](e) induced a pH(i) decrease (-0.51 +/- 0.03 pH units) and impaired pH(i) recovery from butyrate-induced acid load. Moreover, reduction of [Cl(-)](e) abolished the inhibitory effect of foliomycin and markedly reduced the HOE 694- and S3226-sensitive components of pH(i), indicating a role of Cl(-) in the function of these H(+) extrusion mechanisms. We conclude that a vH(+)-ATPase is expressed in ovine REC and plays a considerable role in the pH(i) regulation of these cells.
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PMID:A vH+-ATPase is present in cultured sheep ruminal epithelial cells. 1682 6

The vacuolar-type H(+)-ATPase (V-ATPase) in the plasma membrane of a variety of cells serves as an acid-secreting pathway, and its activity is closely related to cellular functions. Massive proton secretion often leads to electrolyte disturbances in the vicinity of the cell and may in turn affect the activity of the V-ATPase. We characterized, for the first time, the proton currents mediated by plasmalemmal V-ATPase in murine osteoclast-like cells and investigated its activity over a wide range of pH gradients across the membrane (DeltapH = extracellular pH - intracellular pH). The V-ATPase currents were identified as outward H(+) currents and were dependent on ATP and sensitive to the inhibitors bafilomycin A(1) and N,N'-dicyclohexylcarbodiimide. Although H(+) was transported uphill, the electrochemical gradient for H(+) affected the current. The currents were increased by elevating DeltapH and depolarization, and were reduced by lowering DeltapH and hyperpolarization. Elevation of extracellular Ca(2+) (5-40 mm) diminished the currents in a dose-dependent manner and made the voltage dependence more marked. Extracellular Mg(2+) mimicked the inhibition. With 40 mm Ca(2+), the currents decreased to < 40% at 0 mV and to < 10% at about -80 mV. Increases in the intracellular Ca(2+) (0.5-5 microm) did not affect the current. The data suggest that acid secretion through the plasmalemmal V-ATPase is regulated by a combination of the pH gradient, the membrane potential and the extracellular divalent cations. In osteoclasts, the activity-dependent accumulation of acids and Ca(2+) in the closed extracellular compartment might serve as negative feedback signals for regulating the V-ATPase.
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PMID:pH dependence and inhibition by extracellular calcium of proton currents via plasmalemmal vacuolar-type H+-ATPase in murine osteoclasts. 1690 40

Uptake of Na(+) from the environment is an indispensable strategy for the survival of freshwater fish, as they easily lose Na(+) from the plasma to a diluted environment. Nevertheless, the location of and molecules involved in Na(+) uptake remain poorly understood. In this study, we utilized Sodium Green, a Na(+)-dependent fluorescent reagent, to provide direct evidence that Na(+) absorption takes place in a subset of the mitochondria-rich (MR) cells on the yolk sac surface of zebrafish larvae. Combined with immunohistochemistry, we revealed that the Na(+)-absorbing MR cells were exceptionally rich in vacuolar-type H(+)-ATPase (H(+)-ATPase) but moderately rich in Na(+)-K(+)-ATPase. We also addressed the function of foxi3a, a transcription factor that is specifically expressed in the H(+)-ATPase-rich MR cells. When foxi3a was depleted from zebrafish embryos by antisense morpholino oligonucleotide injection, differentiation of the MR cells was completely blocked and Na(+) influx was severely reduced, indicating that MR cells are the primary sites for Na(+) absorption. Additionally, foxi3a expression is initiated at the gastrula stage in the presumptive ectoderm; thus, we propose that foxi3a is a key gene in the control of MR cell differentiation. We also utilized a set of ion transport inhibitors to assess the molecules involved in the process and discuss the observations.
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PMID:Visualization in zebrafish larvae of Na(+) uptake in mitochondria-rich cells whose differentiation is dependent on foxi3a. 1694 87


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