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 the fluorescent dyes sodium-binding-benzofuran-isophthalate and fura-2 cytosolic free sodium concentration ([Na+]i) and cytosolic free calcium concentration ([Ca2+]i) were investigated in intact human platelets in order to characterize the effect of elevated [Ca2+]i on [Na+]i. Spectrofluorometric studies of [Ca2+]i and [Na+]i in intact platelets were done after specific inhibition of endoplasmic Ca-ATPase by thapsigargin. Thapsigargin increased [Ca2+]i and [Na+]i in platelets. Addition of thapsigargin increased [Na+]i from 23.5 +/- 2.9 mM to 51.6 +/- 11.1 mM (mean +/- S.E., P < 0.05). The thapsigargin induced [Na+]i increase was also seen in the absence of extracellular calcium. In the absence of external sodium the thapsigargin induced [Na+]i increase was abolished, indicating that thapsigargin induced [Na+]i increase was due to sodium influx. Thapsigargin induced sodium influx was blocked after administration of NiCl2. The present results support the idea that the filling state of intracellular calcium stores regulate plasma permeability for sodium.
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PMID:Thapsigargin-induced [Ca2+]i increase activates sodium influx in human platelets. 830 96

When expressed alone in fibroblasts, approximately 80% of newly made H2b subunits of the human asialoglycoprotein receptor are retained and degraded in the endoplasmic reticulum (ER), whereas about 20% reaches the plasma membrane (1). Thapsigargin, an inhibitor of the ER Ca2+ ATPase, blocks ER folding of the H1 (2) as well as of the H2b subunit, prevents maturation of H2b, and accelerates ER degradation of newly made H2b. The secretory pathway is normal in thapsigargin-treated cells, as monitored by maturation of the vesicular stomatitis virus G protein. The protease inhibitors TLCK and TPCK block the first step in ER degradation of H2, an endoproteolytic cleavage just exoplasmic to the membrane-spanning domain. In protease inhibitor-treated cells, the approximately 80% of H2b that would normally be degraded remains in the ER; as judged by migration on nonreducing SDS-polyacrylamide gel electrophoresis this H2b is improperly folded. Thus, incorrectly folded H2b is normally subjected to ER degradation. In the presence of thapsigargin H2b cannot fold properly and is degraded within the ER. The preferential ER degradation of misfolded or unfolded membrane proteins demonstrated here, functions as a step in ER quality control.
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PMID:Unfolded H2b asialoglycoprotein receptor subunit polypeptides are selectively degraded within the endoplasmic reticulum. 831 99

We examined whether thapsigargin increases cellular free calcium ([Ca2+]i) and intracellular sodium concentration ([Na+]i) in cultured rat glomerular mesangial cells. 1 x 10(-6) M Thapsigargin increased [Ca2+]i to 244.4 from 86.6 nM, an increase sustained at least during the 15 min observation period. Such an increase in [Ca2+]i was transient in Ca(2+)-free medium containing 1 x 10(-4) M EGTA. An increase in [Ca2+]i by thapsigargin was not altered by 1 x 10(-6) M nicardipine, a L-type Ca2+ channel blocker. Thapsigargin also produced a sustained rise in [Na+]i in a dose-dependent manner. However, preincubation of cells with Ca(2+)-free medium completely blocked the increase in [Na+]i by thapsigargin. These results indicate that thapsigargin increases [Ca2+]i by blocking endoplasmic Ca(2+)-ATPase and enhancing Ca2+ entry, and that the increased Ca2+ influx is triggering an increase in [Na+]i stimulated by thapsigargin per se in glomerular mesangial cells.
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PMID:Thapsigargin increases cellular free calcium and intracellular sodium concentrations in cultured rat glomerular mesangial cells. 833 42

1. The interaction of Ca2+ transport in the plasmalemma and the sarcoplasmic reticulum (SR) was investigated in smooth muscle of the rabbit inferior vena cava. We tested the possibility of direct refilling of the SR with extracellular Ca2+ and of the existence of a vectorial Ca2+ extrusion pathway from the SR lumen to the extracellular space suggested by earlier results. 2. After depletion with caffeine the SR was loaded with Ca2+ to increasing levels by incubation in a high potassium 1.5 mM Ca2+ solution and a 10 mM Ca2+ zero Na+ solution, respectively. Thapsigargin, 2 microM, (a specific SR Ca(2+)-ATPase blocker) completely blocked refilling of the SR in either of the above solutions, indicating that the SR Ca(2+)-ATPase is essential for this process. 3. Three different agents, caffeine, ryanodine and thapsigargin, which inhibit Ca2+ accumulation by the SR, increased the steady state intracellular Ca2+ concentration in the rabbit inferior vena cava. 4. Measurements of Mn2+ induced quenching of the intracellular fura-2 signal during pharmacological manipulation of the SR content showed that these three agents did not stimulate divalent cation entry. 5. On the other hand, stimulation with noradrenaline caused a marked increase in Mn2+ influx, which was blocked by 2 mM Ni2+. Mn2+ entry stimulated by high K+ solution was blocked by 1 microM diltiazem. 6. We conclude that the SR refilling has to be mediated by the SR Ca(2+)-ATPase. Inhibition of Ca2+ accumulation by the SR causes an increase in the steady state intracellular Ca2+ concentration. This observation cannot be explained by an increase in Ca2+ influx into the smooth muscle cells of the rabbit inferior vena cava. Alternatively these results suggest the existence of a continuous vectorial release of Ca2+ from the SR lumen to the extracellular space.
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PMID:The superficial buffer barrier in venous smooth muscle: sarcoplasmic reticulum refilling and unloading. 835 39

1. The goal of this study was to determine if an increase in cytoplasmic calcium concentration ([Ca2+]i), in the absence of additional second messengers derived from membrane phospholipid turnover, is a sufficient signal to induce chloride secretion across monolayers of the human colonic epithelial line, T84. 2. Thapsigargin was used to increase [Ca2+]i by inhibiting the endomembrane Ca(2+)-ATPase. [Ca2+]i was monitored in monolayers by fura-2 fluorescence spectroscopy, chloride secretion by measuring changes in short circuit current (Isc) in modified Ussing chambers, and inositol phosphates were measured by radio-h.p.l.c. of extracts of cells prelabelled with [3H]-inositol. 3. Thapsigargin increased [Ca2+]i and Isc in parallel, without increasing any inositol phosphates. The effect of thapsigargin on Isc was abolished by the intracellular calcium chelator, bis-(o-aminophenoxy)-ethane-N,N,N',N"-tetraacetic acid (BAPTA). 4. Increasing [Ca2+]i with thapsigargin did not prevent a subsequent calcium response to carbachol or histamine if extracellular calcium was available. In the absence of extracellular calcium, only one such release of calcium to hormonal stimulation occurred when cells were pretreated with thapsigargin, and a second response to either carbachol histamine was essentially abolished. 5. Addition of carbachol or histamine to thapsigargin-treated cells mounted in Ussing chambers caused a transient further increase in Isc followed by termination of the response, even though [Ca2+]i continued to rise. 6. We conclude that an elevation in [Ca2+]i is a sufficient signal to induce chloride secretion in T84 cells. Rather than being required to stimulate secretory responses, additional second messengers induced by hormonal secretagogues (such as inositol phosphates) may in fact serve to limit the secretory response.
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PMID:Activation by calcium alone of chloride secretion in T84 epithelial cells. 835 50

The effect of thapsigargin on intracellular free calcium ([Ca2+]i) was examined in swim up human sperm. Thapsigargin elevated [Ca2+]i in a dose dependent manner, with maximal effects observed with 10 microM. The increase in [Ca2+]i was relatively slow in taking 3-4 min to reach a maximal level. The increase in [Ca2+]i was inhibited when extracellular Ca2+ was chelated with EGTA, thus the source of Ca2+ for the increase was extracellular. The effect of thapsigargin to stimulate Ca2+ influx was also observed using the Mn2+ quenching of intracellular Fura-2. Thapsigargin pre-treatment of sperm was able to potentiate the effects of progesterone to elevate [Ca2+]i. Progesterone pre-treatment also potentiated the ability of thapsigargin to elevate [Ca2+]i. Further evidence to support the idea that thapsigargin was promoting Ca2+ influx was the fact that thapsigargin potentiated ionomycin (an agent that can increase Ca2+ cycling across phospholipid membranes) induced elevations in [Ca2+]i. Conversely, ionomycin also potentiated thapsigargin induced elevations in [Ca2+]i. It is concluded from these studies that high concentrations of thapsigargin are able to stimulate Ca2+ influx in human sperm by a mechanism not involving the endoplasmic reticulum Ca(2+)-ATPase pump since this organelle is absent in mature sperm. However, a likely site of action of thapsigargin in mature sperm is at the level of the nuclear membrane Ca(2+)-ATPase pump which is identical to the endoplasmic reticulum Ca(2+)-ATPase pump and is sensitive to inhibition by thapsigargin.
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PMID:Thapsigargin elevates and potentiates the ability of progesterone to increase intracellular free calcium in human sperm: possible role of perinuclear calcium. 838 64

In C6-2B rat glioma cells, agonist-stimulated cAMP accumulation is potently inhibited after the stimulation of endogenous bradykinin receptors or stably transfected substance K receptors, coupled to phosphatidylinositol hydrolysis. In the present report, pharmacological tools were used to selectively stimulate either protein kinase C or Ca2+, the two final effectors activated upon phosphatidylinositol hydrolysis, and their role in the inhibition of the C6-2B cell cAMP signaling pathway was investigated. Activation of protein kinase C by an acute treatment with phorbol 12-myristate 13-acetate or L-alpha-1-oleoyl-2-acetyl-sn-3-glycerol did not reduce, but rather enhanced, the cAMP accumulation elicited by forskolin, a direct activator of adenylyl cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. This effect was antagonized by the protein kinase inhibitor H-7 and mimicked by the protein phosphatase inhibitor okadaic acid. Thapsigargin, a selective microsomal Ca(2+)-ATPase inhibitor, evoked a sustained increase in the intracellular free Ca2+ concentration, with an EC50 of 24.8 +/- 4.3 nM, and inhibited the cAMP accumulation induced by the beta-adrenergic receptor agonist isoproterenol with comparable potency (IC50 = 19.3 +/- 0.2 nM), strongly suggesting a causal relationship between the two phenomena. The inhibition by thapsigargin of isoproterenol- or forskolin-stimulated cAMP accumulation was not affected by pertussis toxin or down-regulation or inhibition of protein kinase C. Dantrolene, a blocker of Ca2+ release from intracellular stores, antagonized 1) the Ca2+ transient in response to thapsigargin and substance K and 2) the inhibitory effect of these compounds on isoproterenol- or forskolin-induced cAMP accumulation. Moreover, sequestration of intracellular Ca2+ with the cell-permeable Ca2+ chelator ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid acetoxymethyl ester abolished the cAMP inhibition mediated by thapsigargin. Finally, isoproterenol- or forskolin-stimulated adenylyl cyclase activity in digitonin-permeabilized cells was not affected by either thapsigargin or substance K. These data provide compelling evidence that increases in intracellular free Ca2+ concentration without activation of protein kinase C suffice and are responsible for the inhibition of cAMP accumulation in C6-2B cells.
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PMID:Ca2+ inhibition of beta-adrenergic receptor- and forskolin-stimulated cAMP accumulation in C6-2B rat glioma cells is independent of protein kinase C. 838 3

In the population of primary cultured rat chromaffin cells, over half exhibited spontaneous [Ca2+]i oscillations, whereas most others were induced to oscillate by low concentrations of bradykinin or KCl. [Ca2+]i spots were observed to pulsate in a defined cytoplasmic area (the oscillator). In silent cells those spots remained discrete, whereas in oscillating cells the [Ca2+]i increase expanded to occupy the entire cytoplasm. Alternation of these discrete and expanded events was observed in a few irregularly oscillating cells. Thapsigargin induced prompt blockade of both pulsations and oscillations and prevented recruitment of silent cells to oscillate. This indicates sarcoendoplasmic reticulum Ca(2+)-ATPase-type Ca2+ pump(s) to be crucial for the functioning of the oscillator. Effects of other treatments were variable, depending on the concomitant [Ca2+]i changes. Oscillations were blocked when EGTA or nitrendipine decreased Ca2+ influx and thus [Ca2+]i; they were also blocked when [Ca2+]i was markedly increased by excess KCl, bradykinin, or ryanodine. When in contrast the [Ca2+]i increases induced by the latter agents remained moderate, oscillations were stimulated. The rhythmic activity of rat chromaffin cells appears, therefore, to operate under a complex regulation that requires [Ca2+]i within an appropriate operative range and does not involve directly the ryanodine receptor but might rely on the activation of IP3 receptors.
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PMID:Mechanism of [Ca2+]i oscillations in rat chromaffin cells. Complex Ca(2+)-dependent regulation of a ryanodine-insensitive oscillator. 839 69

We have investigated plasma membrane Ca2+ transport by monitoring the fluorescence of human peripheral T-lymphocytes loaded with fura 2. Thapsigargin (TG) was utilized the block the Ca(2+)-ATPase of the endoplasmic reticulum and elevate the cytosolic Ca2+ (Ca2+i). Ca2+ influx was inhibited by chelating extracellular Ca2+ with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). The rate of decline in the Ca2+i signal of TG-treated lymphocytes after exposure to EGTA was used to assess Ca2+ extrusion across the plasma membrane. Initial rates of Ca2+i decline were examined in cells suspended in Na(+)-containing and Na(+)-free solutions; initial rates were linearly related to the [Ca2+]i at the onset of the Ca2+i decline and were unaffected by varying the extracellular Ca2+. Extracellular Na+ increased the rate of Ca2+ extrusion and decreased the threshold [Ca2+]i for extrusion, indicating a substantial role for the Na(+)-Ca2+ exchange in Ca2+i homeostasis. Both decreased temperature and calmodulin inhibition significantly slowed the Ca2+i decline in Na(+)-free HEPES-buffered solution, suggesting Ca2+ extrusion under these conditions was mediated by the Ca2+ pump. Protein kinase C (PKC) activation or inhibition did not affect the Ca2+i decline parameters. However, Ca2+ accumulation and Mn2+ (a Ca2+ surrogate) uptake were significantly and Mn2+ (a Ca2+ surrogate) uptake were significantly inhibited by activators of PKC. Cyclic nucleotides altered neither the parameters of the Ca2+i decline nor Mn2+ uptake. Thus human T-lymphocytes exhibit Na(+)- and Ca(2+)-dependent transporters characterized as the Na(+)-Ca2+ exchanger and Ca2+ pump. The main effect of PKC in these cells is the modulation of Ca2+ entry across the lymphocyte plasma membrane.
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PMID:Kinetics of calcium transport across the lymphocyte plasma membrane. 839 24

Thapsigargin, a tumour-promoting sesquiterpene lactone, selectively inhibits the Ca(2+)-ATPase responsible for Ca2+ accumulation by the endoplasmic reticulum (ER). Mobilization of ER-sequestered Ca2+ to the cytosol and to the extracellular fluid subsequently ensues, with concomitant alteration of cellular functions. Thapsigargin was found to serve as a rapid, potent and efficacious inhibitor of amino acid incorporation in cultured mammalian cells. At concentrations mobilizing cell-associated Ca2+ to the extracellular fluid, thapsigargin provoked extensive inhibition of protein synthesis within 10 min. The inhibition in GH3 pituitary cells involved the synthesis of almost all polypeptides, was not associated with increased cytosolic free Ca2+ concentration ([Ca2+]i), and was not reversed at high extracellular Ca2+. The transient rise in [Ca2+]i triggered by ionomycin was diminished by thapsigargin. Polysomes failed to accumulate in the presence of the drug, indicative of impaired translational initiation. With longer (1-3 h) exposures to thapsigargin, recovery of translational activity was observed accompanied by increased synthesis of the ER protein glucose-regulated stress protein 78 or immunoglobulin heavy-chain binding protein ('GRP78/BiP') and its mRNA. Such inductions were comparable with those observed previously with Ca2+ ionophores which mobilize the cation from all intracellular sequestered sites. Actin mRNA concentrations declined significantly during such treatments. In HepG2 cells processing and secretion of the glycoprotein alpha 1-antitrypsin were rapidly suppressed by thapsigargin. Ca2+ sequestered specifically by the ER is concluded to be essential for optimal protein synthesis and processing. These rapid effects of thapsigargin on mRNA translation, protein processing and gene expression should be considered when evaluating potential mechanisms by which this tumour promoter influences cellular events.
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PMID:Inhibition of protein synthesis and early protein processing by thapsigargin in cultured cells. 842 74


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