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)

Using a polyclonal antiserum raised against the inositol 1,4,5-trisphosphate receptor (IP3R) purified from rat cerebellum, we examined the subcellular distribution of IP3R in canine pancreatic homogenates. IP3R was present primarily in a smooth microsomal fraction (low density), a (high density) rough microsomal (RM) fraction previously shown to consist of highly purified rough endoplasmic reticulum (RER) vesicles, and, to a much lesser extent, in an intermediate density microsomal fraction which did not contain markers for RER or plasma membrane. When the RM fraction was subjected to isopycnic centrifugation on sucrose gradients, IP3R equilibrated at high sucrose densities. When ribosomes were extracted from the RM fraction by treatment with puromycin/high salt, IP3R equilibrated at considerably lighter sucrose densities. This shift in density indicated that IP3R which was present in the RM fraction is associated with the RER. Because of a significant amount of IP3R fractionating into the smooth microsomal fraction (which contains plasma membrane, among other "smooth" membranes) and a considerable amount of IP3R present in the nuclear pellet which is also enriched in plasma membrane, we examined the possibility that IP3R may be present in plasma membrane. Further subfractionation of a crude plasma membrane pellet from rat liver revealed that IP3R coenriched with a plasma membrane marker and strongly suggested an association of IP3R with plasma membrane. The issue of why the same receptor is found in multiple biochemically and morphologically distinct membrane fractions is discussed in terms of the possibility of RER subcompartmentalization and IP3R subtypes. The fractionation pattern of IP3R in pancreas is significantly different from that previously reported for calcium (Ca2+)-binding proteins and an intracellular Ca-ATPase (Nigam, S. K. and Towers, T. (1990) J. Cell Biol. 111, 197-200), raising questions as to links between these latter proteins and IP3 sensitive Ca2+ pools. Nevertheless, although the fractionation patterns are different, all of these proteins are clearly associated with the RER.
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PMID:Inositol 1,4,5-trisphosphate receptors. Localization in epithelial tissue. 131 1

The molecular composition of intracellular Ca2+ stores in developing chicken cerebellum Purkinje neurons from embryonic day 11 (E11) to posthatching day 2 (P2) was studied by immunocytochemistry using specific antibodies for three molecular constituents, the receptor (R) and/or channel sensitive to inositol 1,4,5-trisphosphate (IP3), Ca(2+)-adenosinetriphosphatase (ATPase), and calsequestrin (CS). CS, IP3R, and Ca(2+)-ATPase were first detected by light-microscopic immunofluorescence in migrating Purkinje cells at E11-E12 and throughout late phases of embryonic development. Ontogenesis of CS, IP3R, and Ca(2+)-ATPase accompanied well-defined stages of cerebellum histogenesis and cytogenesis and was accomplished before hatching. High-resolution immunogold electronmicroscopy revealed that, at E18-P1, CS was still largely distributed to the endoplasmic reticulum (ER) lumen and began to be segregated to ER subcompartments (calciosomes) only by P2, whereas the IP3R was concentrated into ER cisternal stacks as early as E18. Both ionotropic and metabotropic plasma membrane receptors were present in dissociated single chicken Purkinje cells from E16 onward, as indicated by measurements of membrane currents (whole cell recording mode) and of cytoplasmic Ca2+ transients monitored with the cell-trappable fluorescent indicator fura 2-acetoxymethyl ester, respectively. Cytoplasmic Ca2+ transients were detected after either activation of glutamate metabotropic receptors, i.e., evidence of IP3-sensitive Ca2+ channels, or application of caffeine, i.e., evidence of ryanodine-sensitive Ca2+ channels. Intracellular Ca2+ stores appear to be functional during embryonic development.
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PMID:Intracellular Ca2+ stores in chick cerebellum Purkinje neurons: ontogenetic and functional studies. 749 12

In an earlier subcellular fractionation study of epithelial tissue (liver and pancreas), we demonstrated that the inositol 1,4,5-trisphosphate receptor (IP3R) is found in association with biochemically distinct cellular membranes, including the endoplasmic reticulum (ER) and plasma membrane (Sharp, A. H., Snyder, S. H., and Nigam, S. K. (1992) J. Biol. Chem. 267, 7444-7449). To further characterize epithelial IP3Rs, we have now employed cultured Madin-Darby canine kidney (MDCK) cells, a well studied tight polarized epithelial cell type. Indirect immunofluorescence with an antiserum which specifically recognizes IP3R in MDCK cells by immunoblotting and immunoprecipitation gave an ER-like staining pattern as well as a basolateral plasma membrane-like staining pattern, the latter being particularly evident in highly confluent monolayers. In sections of adult rat kidney tubules a similar staining pattern was observed. Interestingly, whereas known basolateral proteins (Na+,K(+)-ATPase and the facilitated glucose transporter) gave a continuous basolateral staining pattern, that seen for IP3R was discontinuous (punctate). A highly similar staining pattern was observed for the caveolar protein, caveolin, suggesting that the punctate basolateral plasma membrane-like staining pattern observed for IP3R reflects its localization to basolateral caveolae. Biotinylation of non-permeabilized and permeabilized MDCK cells, followed by immunoprecipitation of IP3R and detection with streptavidin, indicated that while most IP3R is localized to biotin-inaccessible compartments (i.e. ER), a fraction (10-20%) of IP3R was accessible to externally added biotin primarily from the basolateral side. This result is compatible with the dual ER and basolateral caveolar localization suggested by immunocytochemistry, although it does not exclude the presence of some IP3R in the basolateral plasma membrane as well. Solubility studies revealed IP3R to be considerably more insoluble than the basolateral proteins, Na+,K(+)-ATPase and the liver cell adhesion molecule, as well as the cytoskeletal proteins, ankyrin and fodrin. In the most insoluble fraction, IP3R was found along with caveolin, further supporting the notion that part of the cellular IP3R pool associates with caveolae. Since multiple localizations of IP3R within a cell might reflect the existence of multiple isoforms, polymerase chain reaction amplification of first strand cDNA with primers specific for the three isotypes of IP3R was performed. All three isoforms of IP3R were expressed in the homogeneous population of MDCK cells. The existence of distinct membrane localizations and multiple isoforms of IP3R within the same cell type suggests an explanation for the complex spatiotemporal patterns of Ca2+ release from inositol 1,4,5-trisphosphate-sensitive Ca2+ pools in epithelial and other cells.
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PMID:Epithelial inositol 1,4,5-trisphosphate receptors. Multiplicity of localization, solubility, and isoforms. 808 40

We undertake a quantitative investigation of changes in intracellular free Ca2+ concentration ([Ca2+]i) in antigen-stimulated rat basophilic leukemia (RBL-2H3) cells, which include contributions of both Ca2+ store release and Ca2+ influx from the medium. Following Keizer and De Young (J. Keizer and G. De Young. Biophys. J. 61: 649-660, 1992), we develop a highly constrained mathematical model for [Ca2+]i oscillations in RBL-2H3 cells, which includes activation of the inositol trisphosphate receptor (IP3R) by inositol 1,4,5-trisphospate, indirect Ca2+ activation of the IP3R via Ca2+ -dependent activity of phospholipase C-gamma, slow inhibition of the IP3R by cytosolic Ca2+, refilling of Ca2+ stores by a Ca2+ -ATPase (SERCA)-type pump, and a simple representation of the dependence of plasma membrane (PM) fluxes on experimental conditions. Using this full (open cell) model, we simulate [Ca2+]i responses for protocols in which antigen concentration and external Ca2+ are manipulated and compare out calculations with experimental data. In protocol A, cells are stimulated in the presence of external Ca2+, in protocols B and C, cells are stimulated in the absence of external Ca2+, with external Ca2+ later reapplied in protocol C. We are able to reproduce quantitatively the important features of all three protocols, including the dose response of protocol B, the [Ca2+]i response to thapsigargin, and lag time results, and we provide qualitative explanations for the responses derived from our calculations. We also develop a simplified (closed cell) version of the model in which PM fluxes are neglected and total free Ca2+ concentration ([Ca2+]T) is a slowly varying parameter. This permits us to explain in a simple graphical fashion how PM fluxes may influence [Ca2+]i responses in RBH-2H3 cells through modulation of [Ca2+]T.
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PMID:Effect of Ca2+ influx on intracellular free Ca2+ responses in antigen-stimulated RBL-2H3 cells. 863 49

The role of reversible phosphorylation in histamine-induced Ca2+ oscillations in HeLa cells has been investigated by using various activators and inhibitors of protein kinases and phosphatases. Electroporation was employed to introduce impermeable materials into single cells, which proved to be a useful and convenient tool. Of the kinases examined, cAMP-dependent kinase, protein kinase C, and calmodulin-dependent kinase II (CaMK II), only CaMK II was essential. When added during oscillations, both W-7, a calmodulin antagonist, and KN-62, a specific CaMK II inhibitor, caused one large Ca2+ spike before halting the process. Introduction of the Ca2+/calmodulin-independent catalytic domain of CaMK II into the cells forestalled their response to histamine. These results show that intracellular Ca2+ cannot oscillate when CaMK II is locked in either the inactive or the stimulated state. External Ca2+ electroporated into cells preloaded with the catalytic domains was quickly removed (but not when the cells were pretreated with the endoplasmic reticulum Ca(2+)-ATPase inhibitor, tapsigargin), indicating that the ATP-driven Ca2+ pump was somehow activated by CaMK II. Protein phosphatase inhibitors calyculin A and okadaic acid abolished ongoing oscillations and, when added at low concentrations, prolonged the interspike interval. Immunoprecipitation experiments with 32P(i)-labeled cells provided the first evidence that inositol 1,4,5-trisphosphate receptor (IP3R) was phosphorylated by CaMK II in vivo. The extent of phosphorylation was increased in the presence of histamine, significantly enhanced by calyculin A, and greatly reduced by W-7. Our observations are consistent with the concept that repetitive phosphorylation-dephosphorylation cycles regulating IP3R and Ca2+ pumps are a controlling factor for sustained Ca2+ oscillations in HeLa, and possibly other, cells.
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PMID:Reversible phosphorylation as a controlling factor for sustaining calcium oscillations in HeLa cells: Involvement of calmodulin-dependent kinase II and a calyculin A-inhibitable phosphatase. 867 50

The inositol 1,4,5-trisphosphate receptor (IP3R) is an endoplasmic reticular calcium release channel found in most cell types. Calcium signaling mediated by IP3Rs regulates a wide variety of physiological processes, including smooth muscle contraction, immune function, and fertility. We have focused on the role of the IP3R in programmed cell death and the regulation of IP3R levels in heart failure, a condition shown to be associated with cardiomyocyte apoptosis. During end-stage human heart failure, we have demonstrated that type 1 IP3R (IP3R1) mRNA and protein levels are up-regulated, in contrast to other cardiac calcium regulatory proteins, such as the type 2 ryanodine receptor (RYR2) and type IIa sarcoplasmic reticulum calcium adenosine triphosphatase (SERCA2), which are down-regulated. These data suggest that altered calcium channel expression may contribute to the defects in calcium homeostasis during heart failure. Furthermore, regulation of the IP3R may have implications for the survival of cardiac myocytes. Data from our laboratory have linked IP3R expression with susceptibility to apoptosis. IP3R-deficient T cells are resistant to apoptosis induced by dexamethasone, T cell receptor stimulation, ionizing radiation, and Fas. These findings suggest that intracellular calcium release via IP3Rs is a critical mediator of apoptosis. Thus the IP3R, which is up-regulated during human heart failure, may play a role in cardiomyocyte apoptosis and therefore in the pathophysiology of heart failure.
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PMID:Role of inositol 1,4,5-trisphosphate receptors in regulating apoptotic signaling and heart failure. 947 44

Expression patterns of sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase (SERCA) and inositol 1,4,5-trisphosphate receptor (IP3R) isoforms were studied in endothelial cells at the mRNA level by ratio RT-PCR technique and subsequent restriction-enzyme analysis. Three types of cells have been used in the present study: rat adrenal medulla microvascular endothelial cells (RAMEC), rat aortic endothelial cells (RAEC), and human umbilical vein endothelial cells (HUVEC). Our data show the presence of multiple SERCA and IP3R isoforms in each type of endothelial cells. Freshly isolated HUVEC were an exception in this respect since they contained only SERCA3 without SERCA2b messengers. The expression patterns changed upon cell proliferation: SERCA3 and IP3R-1 messengers decreased, while IP3R-3 increased with culturing. Upon cell differentiation, induced by culturing the cells on Matrigel, the expression pattern of the IP3R changed even further in all endothelial cell types: IP3R-1 was reduced in all three cell kinds, while IP3R-3 raised significantly in RAEC and RAMEC. In HUVEC the expression of SERCA returned, upon differentiation, to the levels observed in the freshly isolated cells. Thus, the plasticity of expression of various SERCA and IP3R isoforms shows that possibly different Ca2+ pools may play distinct roles in cell proliferation and differentiation.
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PMID:Expression patterns of sarco/endoplasmic reticulum Ca(2+)-ATPase and inositol 1,4,5-trisphosphate receptor isoforms in vascular endothelial cells. 1046 1

The physiological activity of osteoblasts is known to be closely related to increased intracellular Ca2+ activity ([Ca2+]i) in osteoblasts. The cellular regulation of [Ca2+]i in osteoblasts is mediated by Ca2+ movements associated with Ca2+ release from intracellular Ca2+ stores, and transmembrane Ca2+ influx via Na+-Ca2+ exchanger, and Ca2+ ATPase. Reactive oxygen species, such as H2O2, play an important role in the regulation of cellular functions, and act as signaling molecules or toxins in cells. In this study, we investigated the effects of H2O2 on cellular Ca2+ regulation in osteoblasts by measuring intracellular Ca2+ activities using cellular calcium imaging techniques. Osteoblasts were isolated from the femurs and tibias of neonatal rats, and cultured for 7 days. The cultured osteoblasts were loaded with a Ca2+-sensitive fluorescent dye, Fura-2, and fluorescence images were monitored using a cooled CCD camera, and subsequently analyzed using image analyzing software. The results obtained are as follows: (1) The osteoblasts with lower basal Ca2+ activities yielded a transient Ca2+ increase, a Ca2+ spike, while osteoblasts with higher basal Ca2+ activities showed a continuous increase in [Ca2+]i leading to cell death. (2) Ca2+ spikes, generated after removing Na+ from superfusing solutions, were blocked by H2O2 and this was followed by a sustained increase in Ca2+ activity. (3) ATP- induced Ca2+ spikes were inhibited by pretreating with H2O2 and this was followed by a continuous increase of [Ca2+]i. When cells were pretreated with the exogenous nitric oxide (NO) donor S-Nitroso-N-acetylpenicilance (SNAP, 50 microM), treatments of ATP (1 mM) induced a Ca2+ spike-like increase, but [Ca2+]i did not return to the basal level. (4) The expression of inositol- 1,4,5-triphosphate receptor (IP3R) was enhanced by H2O2. Our results suggest that H2O2 modulates intracellular Ca2+ activity in osteoblasts by increasing Ca2+ release from the intracellular Ca2+ stores.
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PMID:H2O2 enhances Ca2+ release from osteoblast internal stores. 1197 Dec 17

It now generally is agreed that Na,K-ATPase, in addition to its role in the maintenance of Na+ and K+ gradients across the cell membrane, plays a role in communicating information from the extracellular environment to intracellular signaling pathways. It was reported recently that interaction between ouabain-bound Na,K-ATPase and the 1,4,5-trisphosphate receptor (IP3R) triggers slow calcium oscillations and activation of NF-kappaB. Here it is demonstrated that this signaling pathway can serve to prevent cell death and promote cell growth. Rat renal proximal tubular cells in primary culture first were grown in the presence of 10% serum and then exposed to 0.2% serum for 24 h to induce apoptosis. Serum starvation increased the apoptotic index from 1.21 +/- 0.26 to 14.01 +/- 1.17%. Ouabain in concentrations that did not inhibit Na,K-ATPase activity (1 to 10 nM) completely abolished the apoptotic effect of serum starvation. Ouabain protection from apoptosis was not observed when release of calcium from intracellular stores via the IP3R was prevented. It was shown that the NH2 terminal tail of the Na,K-ATPase alpha subunit plays a key role in ouabain-triggered calcium oscillations. It was found that ouabain did not protect from apoptosis in serum-deprived cells that expressed a mutant Na,K-ATPase alpha subunit with deletion of the NH2 terminal tail. Ouabain exposure (10 nM for 24 h) significantly increased translocation of NF-kappaB from cytoplasm to nucleus. Helenalin, an inhibitor of NF-kappaB, abolished the antiapoptotic effect of ouabain. Ouabain (0.1 to 10 nM) also was found to stimulate proliferation and increase the viability of kidney cells. These effects were abolished when release of calcium via the IP3R was prevented.
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PMID:Low doses of ouabain protect from serum deprivation-triggered apoptosis and stimulate kidney cell proliferation via activation of NF-kappaB. 1670 66

The mitochondrial calcium and downstream proline-rich tyrosine kinase-2 (PyK2) signaling pathway are critical to hepatitis B virus (HBV) replication, and the endoplasmic reticulum (ER) plays an important role in intracellular calcium regulation. To investigate the role of ER in HBV replication, the HBV genome transfected HepG2.2.15 cells were treated by cyclosporine A (CsA), cyclopiazonic acid (CPA), ryanodine and U73122, which are all specific blockers of calcium channels located in either ER or mitochondria. The HBV replication level was evaluated by two methods: slot blot hybridization analysis of intracellular HBV DNA and real-time polymerase chain reaction (PCR) analysis of secreted HBV DNA in supernatant; the activation of PyK2 kinase was detected by Western blot analysis. Results indicated that the HBV replication was inhibited when mitochondrial permeability transition pore, ER Ca2+ -ATPase and ER inositol 1,4,5-trisphosphate receptor (IP3R) were blocked by CsA, CPA and U73122, respectively; but not inhibited when ER ryanodine receptor was blocked by ryanodine. The PyK2 phosphorylation level declined after treatment of 2 microg/ml CsA, 5 microM CPA and 25 microM U73122, but not changed apparently after 50 microM ryanodine treatment. Compared with monotreatment, a more powerful inhibitory effect was achieved when the CsA, CPA and U73122 were combined used in twosome or triple manner, while the HBV replication level did not change apparently when ryanodine combined with CsA, CPA or U73122. In conclusion, besides the mitochondria, the ER also participates in the HBV replication through calcium-PyK2 signaling pathway; the calcium channels of ER Ca2+ -ATPase and ER IP3R are responsible for this role; during this complicated process, an interaction between ER and mitochondria maybe involved.
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PMID:Involvement of endoplasmic reticulum in hepatitis B virus replication. 1687 Feb 95


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