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)

1. Cibenzoline, (+/-)-2-(2,2-diphenylcyclopropyl-2-imidazoline succinate, has been clinically used as one of the Class I type antiarrhythmic agents and also reported to block ATP-sensitive K(+) channels in excised membranes from heart and pancreatic beta cells. In the present study, we investigated if this drug inhibited gastric H(+),K(+)-ATPase activity in vitro. 2. Cibenzoline inhibited H(+),K(+)-ATPase activity of permeabilized leaky hog gastric vesicles in a concentration-dependent manner (IC(50): 201 microM), whereas no effect was shown on Na(+),K(+)-ATPase activity of dog kidney (IC(50): >1000 microM). Similarly, cibenzoline inhibited H(+),K(+)-ATPase activity of HEK-293 cells (human embryonic kidney cell line) co-transfected with rabbit gastric H(+),K(+)-ATPase alpha- and beta-subunit cDNAs (IC(50): 183 microM). 3. In leaky gastric vesicles, inhibition of H(+),K(+)-ATPase activity by cibenzoline was attenuated by the addition of K(+) (0.5 - 5 mM) in a concentration-dependent manner. The Lineweaver-Burk plot of the H(+),K(+)-ATPase activity shows that cibenzoline increases K(m) value for K(+) without affecting V(max), indicating that this drug inhibits H(+),K(+)-ATPase activity competitively with respect to K(+). 4. The inhibitory effect of H(+),K(+)-ATPase activity by cibenzoline with normal tight gastric vesicles did not significantly differ from that with permeabilized leaky gastric vesicles, indicating that this drug reacted to the ATPase from the cytoplasmic side of the membrane. 5. These findings suggest that cibenzoline is an inhibitor of gastric H(+),K(+)-ATPase with a novel inhibition mechanism, which inhibits gastric H(+),K(+)-ATPase by binding its K(+)-recognition site from the cytoplasmic side.
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PMID:Cibenzoline, an ATP-sensitive K(+) channel blocker, binds to the K(+)-binding site from the cytoplasmic side of gastric H(+),K(+)-ATPase. 1173 41

Sarcolipin (SLN), a regulator of the sarco(endo)plasmic reticulum Ca(2+)-ATPase of fast-twitch skeletal muscle (SERCA1a), is also expressed in cardiac and slow-twitch skeletal muscles where phospholamban (PLN) and SERCA2a are expressed. Co-expression in HEK-293 cells of SLN tagged N-terminally with a FLAG epitope (NF-SLN), PLN, and SERCAs followed by measurement of the Ca(2+) dependence of Ca(2+) transport activity in isolated microsomal fractions showed that NF-SLN can reduce the apparent Ca(2+) affinity of both SERCA1a (DeltaK(Ca) = -0.22 +/- 0.01 pCa units) and SERCA2a (DeltaK(Ca) = -0.37 +/- 0.04 pCa units). When SERCA1a or SERCA2a were co-expressed with both NF-SLN and PLN, inhibition was synergistic, reducing DeltaK(Ca) by about -1.0 pCa units. Co-immunoprecipitation showed that NF-SLN increased the binding of PLN to SERCA, whereas PLN did not increase the binding of NF-SLN to SERCA. Elevated Ca(2+) dissociates both PLN and NF-SLN from their complexes with both SERCA1a and SERCA2a, but NF-SLN induced resistance to Ca(2+) dissociation of the PLN.SERCA complex. Co-immunoprecipitation of PLN and NF-SLN without SERCA showed that NF-SLN binds directly to PLN and that NF-SLN inhibits the formation of PLN pentamers. Thus the ability of NF-SLN to elevate the content of PLN monomers can account, at least in part, for the superinhibitory effects of NF-SLN in the presence of PLN.
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PMID:Sarcolipin inhibits polymerization of phospholamban to induce superinhibition of sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs). 1203 37

Analysis of small dorsal root ganglion (DRG) neurons revealed novel functions for vanilloid receptor 1 (VR1) in the regulation of cytosolic Ca(2+). The VR1 agonist capsaicin induced Ca(2+) mobilization from intracellular stores in the absence of extracellular Ca(2+), and this release was inhibited by the VR1 antagonist capsazepine but was unaffected by the phospholipase C inhibitor xestospongins, indicating that Ca(2+) mobilization was dependent on capsaicin receptor binding and was not due to intracellular inositol-1,4,5-trisphosphate generation. Confocal microscopy revealed extensive expression of VR1 on endoplasmic reticulum, consistent with VR1 operating as a Ca(2+) release receptor. The main part of the capsaicin-releasable Ca(2+) store was insensitive to thapsigargin, a selective endoplasmic reticulum Ca(2+)-ATPase inhibitor, suggesting that VR1 might be predominantly localized to a thapsigargin-insensitive endoplasmic reticulum Ca(2+) store. In addition, VR1 was observed to behave as a store-operated Ca(2+) influx channel. In DRG neurons, capsazepine attenuated Ca(2+) influx following thapsigargin-induced Ca(2+) store depletion and inhibited thapsigargin-induced inward currents. Conversely, transfected HEK-293 cells expressing VR1 showed enhanced Ca(2+) influx and inward currents following Ca(2+) store depletion. Combined data support topographical and functional diversity for VR1 in the regulation of cytosolic Ca(2+) with the plasma membrane-associated form behaving as a store-operated Ca(2+) influx channel and endoplasmic reticulum-associated VR1 possibly functioning as a Ca(2+) release receptor in sensory neurons.
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PMID:Versatile regulation of cytosolic Ca2+ by vanilloid receptor I in rat dorsal root ganglion neurons. 1245 15

Characteristics of the cellular response to oxygen deprivation and subsequent reoxygenation (hypoxia/reoxygenation) include redirection of energy metabolism, increased glucose utilization and expression of oxygen-regulated proteins. Inhibition of protein synthesis during early reoxygenation period prevented effective astrocyte adaptation to hypoxia/reoxygenation, resulting in eventual cell death. To elucidate the role of astrocytes in the central nervous system in response to hypoxia/reoxygenation, we analyzed the cDNA library derived from the cultured rat astrocytes subjected to 24 h of hypoxia followed by reoxygenation by differential display, and isolated a cDNA corresponding to Na/K ATPase alpha1 subunit. The expression of Na/K ATPase alpha1 subunit mRNA as well as beta1subunit mRNA was transiently increased after reoxygenation, whereas hypoxia itself did not induce any gene expression change. Na/K ATPase alpha1 subunit protein was transiently increased, whereas the protein expression for Na/K ATPase beta1 subunit showed sustained induction after reoxygenation. Overexpression of beta1 subunit in HEK 293 cells subjected to hypoxia/reoxygenation promoted survival of the cells. These findings suggest that Na/K ATPases may contribute to maintain the cellular environment of astrocytes subjected to hypoxia/reoxygenation.
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PMID:Induction of mRNAs and proteins for Na/K ATPase alpha1 and beta1 subunits following hypoxia/reoxygenation in astrocytes. 1257 31

The influences of the gastric H+/K+ pump on organelle pH during trafficking to and from the plasma membrane were investigated using HEK-293 cells stably expressing the alpha- and beta-subunits of human H+/K+-ATPase (H+/K+-alpha,beta cells). The pH values of trans-Golgi network (pHTGN) and recycling endosomes (pHRE) were measured by transfecting H+/K+-alpha,beta cells with the pH-sensitive GFP pHluorin fused to targeting sequences of either TGN38 or synaptobrevin, respectively. Immunofluorescence showed that H+/K+-ATPase was present in the plasma membrane, TGN, and RE. The pHTGN was similar in both H+/K+-alpha,beta cells (pHTGN 6.36) and vector-transfected ("mock") cells (pHTGN 6.34); pHRE was also similar in H+/K+-alpha,beta (pHRE 6.40) and mock cells (pHRE 6.37). SCH28080 (inhibits H+/K+-ATPase) caused TGN to alkalinize by 0.12 pH units; subsequent addition of bafilomycin (inhibits H+ v-ATPase) caused TGN to alkalinize from pH 6.4 up to a new steady-state pHTGN of 7.0-7.5, close to pHcytosol. Similar results were observed in RE. Thus H+/K+-ATPases that trafficked to the plasma membrane were active but had small effects to acidify the TGN and RE compared with H+ v-ATPase. Mathematical modeling predicted a large number of H+ v-ATPases (8000) active in the TGN to balance a large, passive H+ leak (with PH approximately 10-3 cm/s) via unidentified pathways out of the TGN. We propose that in the presence of this effective, though inefficient, buffer system in the Golgi and TGN, H+/K+-ATPases (estimated to be approximately 4000 active in the TGN) and other transporters have little effect on luminal pH as they traffic to the plasma membrane.
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PMID:pH of TGN and recycling endosomes of H+/K+-ATPase-transfected HEK-293 cells: implications for pH regulation in the secretory pathway. 1266 Jan 45

Outer nuclear membrane is endowed with a SERCA type Ca(2+)-ATPase which pumps calcium into the nuclear envelope lumen and creates calcium stores. Variation in this calcium pool, among other things, regulates nuclear transport. The transport of Nuclear Localization Signal (NLS)-containing molecules into the nucleus is well established. Intermediate size molecules lacking an NLS translocate to the nucleus and its mechanism remains obscure. It is observed here that the treatment of HEK 293 cells in culture with dibutyryl cyclic AMP (db-cAMP) or forskolin (FK) triggered transport of Calcium Green 10 kDa dextran into the nucleus. Under similar conditions Fluo-3-AM accumulated around the nuclei. cAMP-dependent protein kinase phosphorylated 105 kDa nuclear Ca(2+)-ATPase (NCA) which served as a trigger for NLS-independent transport into the nucleus.
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PMID:In vivo nuclear Ca2+-ATPase phosphorylation triggers intermediate size molecular transport to the nucleus. 1268 66

The high risk forms of human papillomavirus (HPV) (primarily types 16 and 18) are the leading cause of cervical cancer worldwide. Infection results in expression of three oncoproteins, E5, E6, and E7, the latter two being of predominant importance in maintaining a transformed state of the host epithelial cell. While little is known about the role(s) of the HPV E5, the bovine papillomavirus type 1 (BPV1) E5 protein has been well characterized. A study of HPV16 E5 was performed, focusing on the protein's ability to self-interact, its ability to bind to the 16-kDa subunit of the vacuolar H(+)-ATPase (16K), and its cellular localization. As has been previously shown for BPV1 E5, we found that HPV16 E5 is also capable of self-interaction and binding to 16K. Further, we examined which portions of the HPV16 E5 protein were involved in these interactions using progressive deletions of putative transmembrane helices of the protein. All of the E5 deletion mutants tested bound to full-length E5 as well as to 16K, suggesting that these protein-protein interactions are based on hydrophobic interactions. The majority of E5 expressed in HEK 293-T7 cells was perinuclear but did not appear to localize to the cis/medial-Golgi, in contrast to previous reports for both HPV16 E5 and BPV1 E5.
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PMID:Oligomerization of the E5 protein of human papillomavirus type 16 occurs through multiple hydrophobic regions. 1295 9

Increasing hypoxia tolerance in mammalian cells is potentially of major importance, but it has not been feasible thus far. The disaccharide trehalose, which accumulates dramatically during heat shock, enhances thermotolerance and reduces aggregation of denatured proteins. Previous studies from our laboratory showed that over-expression of Drosophila trehalose-phosphate synthase (dtps1) increases the trehalose level and anoxia tolerance in flies. To determine whether trehalose can protect against anoxic injury in mammalian cells, we transfected the dtps1 gene into human HEK-293 cells using the recombinant plasmid pcDNA3.1(-)-dtps1 and obtained more than 20 stable cell strains. Glucose starvation in culture showed that HEK-293 cells transfected with pcDNA3.1(-)-dtps1 (HEK-dtps1) do not metabolize intracellular trehalose, and, interestingly, these cells accumulated intracellular trehalose during hypoxic exposure. In contrast to HEK-293 cells transfected with pcDNA3.1(-) (HEK-v), cells with trehalose were more resistant to low oxygen stress (1% O2). To elucidate how trehalose protects cells from anoxic injury, we assayed protein solubility and the amount of ubiquitinated proteins. There was three times more insoluble protein in HEK-v than in HEK-dtps1 after 3 days of exposure to low O2. The amount of Na+-K+ ATPase present in the insoluble proteins dramatically increased in HEK-v cells after 2 and 3 days of exposure, whereas there was no significant change in HEK-dtps1 cells. Ubiquitinated proteins increased dramatically in HEK-v cells after 2 and 3 days of exposure but not in HEK-dtps1 cells over the same period. Our results indicate that increased trehalose in mammalian cells following transfection by the Drosophila tps1 gene protects cells from hypoxic injury. The mechanism of this protection is likely related to a decrease in protein denaturation, through protein-trehalose interactions, resulting in enhanced cellular recovery from hypoxic stress.
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PMID:Expression of Drosophila trehalose-phosphate synthase in HEK-293 cells increases hypoxia tolerance. 1312 20

Mammalian cells regulate their cytosolic pH through a variety of proton extruding and bicarbonate loading mechanisms. The human embryonic kidney cell line HEK 293 is thought to show some characteristics of proximal tubule cells. The present study was performed to investigate the activity of proton extruding mechanisms (i.e. Na(+)/H(+) exchange and/or V-H(+)-ATPases) and the influence of intracellular pH (pH(i)) on the activation of these transport processes. At resting pH(i) (7.4) and in the absence of bicarbonate, removal of extracellular Na(+) did not alter pH(i) Intracellular acidification (pH(i) approximately 6.2) after a NH(4)Cl prepulse (20 mM) followed by exposure to a Na(+) free bath solution led to a slow pH(i) recovery (with a delay of 5 min), which was inhibited by the specific vacuolar H(+)-ATPase inhibitor bafilomyocin. There was no Na(+)-dependent pH(i) recovery upon exposure to Na(+) after a short intracellular acidification (less than 5 min). However, when the intracellular acidification phase was extended, the activation of a Na(+)-dependent pH(i) recovery was seen. This Na(+)-dependent pH(i) recovery was inhibited by 30 microM EIPA but not by 2 microM EIPA or less, suggesting the involvement of NHE3. Western blot analysis confirmed the presence of NHE3 protein in HEK 293 cells. Disruption of the microtubular network by colchicine (20 microM) did not significantly inhibit the rate of Na(+)-independent or dependent pH(i) recovery indicating that activation of both H(+)-ATPase, and the NHE were not due to stimulated trafficking of the transport proteins or some activators to the membrane. We conclude that a) the plasma membrane of HEK293 cells contains both, NHE and H(+)-ATPase and b) both H(+) extrusion systems are inactive at neutral pH(i), c) a decrease of cytosolic pH to 6.5 activates both transport proteins in a slow, time-dependent manner.
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PMID:Intracellular pH activates membrane-bound Na(+)/H(+) exchanger and vacuolar H(+)-ATPase in human embryonic kidney (HEK) cells. 1458 69

Sarcolipin (SLN) inhibits the cardiac sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA2a) by direct binding and is superinhibitory if it binds through phospholamban (PLN). To determine whether overexpression of SLN in the heart might impair cardiac function, transgenic (TG) mice were generated with cardiac-specific overexpression of NF-SLN (SLN tagged at its N terminus with the FLAG epitope). The level of NF-SLN expression (the NF-SLN/PLN expression ratio) was equivalent to that which induces profound superinhibition when coexpressed with PLN and SERCA2a in HEK-293 cells. In TG hearts, the apparent affinity of SERCA2a for Ca(2+) was decreased compared with non-TG littermate control hearts. Invasive hemodynamic and echocardiographic analyses revealed impaired cardiac contractility and ventricular hypertrophy in TG mice. Basal PLN phosphorylation was reduced. In isolated papillary muscle subjected to isometric tension, peak amplitudes of Ca(2+) transients and peak tensions were reduced, whereas decay times of Ca(2+) transients and relaxation times of tension were increased in TG mice. Isoproterenol largely restored contractility in papillary muscle and stimulated PLN phosphorylation to wild-type levels in intact hearts. No compensatory changes in expression of SERCA2a, PLN, ryanodine receptor, and calsequestrin were observed in TG hearts. Coimmunoprecipitation indicated that overexpressed NF-SLN was bound to both SERCA2a and PLN, forming a ternary complex. These data suggest that NF-SLN overexpression inhibits SERCA2a through stabilization of SERCA2a-PLN interaction in the absence of PLN phosphorylation and through the inhibition of PLN phosphorylation. Inhibition of SERCA2a impairs contractility and calcium cycling, but responsiveness to beta-adrenergic agonists may prevent progression to heart failure.
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PMID:Cardiac-specific overexpression of sarcolipin inhibits sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA2a) activity and impairs cardiac function in mice. 1520 33


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