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 effect of thapsigargin on the activity of various enzymes involved in the Ca(2+)-homeostasis of cardiac muscle and on the contractile activity of isolated cardiomyocytes was investigated. Thapsigargin was found to be a potent and specific inhibitor of the Ca(2+)-pump of striated muscle SR (IC50 in the low nanomolar range). A strong reduction of the Vmax of the Ca(2+)-pump was observed while the Km (Ca2+) was only slightly affected. Reduction of the Vmax was caused by the inability of the ATPase to form the Ca(2+)-dependent acylphosphate intermediate. Thapsigargin did not change the passive permeability characteristics nor the function of the Ca(2+)-release channels of the cisternal compartments of the SR. In addition, no significant effects of thapsigargin on other ATPases, such as the Ca(2+)-ATPase and the Na+/K(+)-ATPase of the plasma membrane as well as the actomyosin ATPase could be detected. The contractile activity of paced adult rat cardiomyocytes was completely abolished by 300 nM thapsigargin. At lower concentrations the drug prolonged considerably the contraction-relaxation cycle, in particular the relaxation phase. The intracellular Ca(2+)-transients elicited by electrical stimulation (as measured by the changes in Fluo-3 fluorescence) decreased in parallel and the time needed to lower free Ca2+ down to the resting level increased. In conclusion, the results indicate that selective inhibition of the Ca(2+)-pump of the SR by thapsigargin accounts for the functional degeneration of myocytes treated with the drug.
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PMID:Effect of thapsigargin on cardiac muscle cells. 132 Apr 56

Cai in cultured human sweat gland epithelial monolayers was measured using Fura-2 fluorescence. Thapsigargin (Tg) caused a sustained increase in Cai, the rate of rise being slower but the magnitude greater than with the agonists lysylbradykinin and ATP. Tg caused an irreversible change such that even after it was removed Cai was dependent on the ambient calcium concentration, consistent with the hypothesis that Ca2+ entry is controlled by the state of the intracellular stores. Calcium entry after Tg was not modified by nimodipine, omega-conotoxin, or BAY K8644 but could be blocked by low concentrations (0.5 mM) of La3+. High concentrations of La3+ (2 mM) caused an increase in the response to Tg, suggesting that membrane ATPase exerts a major Cai lowering effect. Intracellular Ca2+ ion chelation with 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid significantly blunted the response to Tg. Finally, Mn2+ entry rate into epithelial cells was doubled by Tg. In spite of the evidence that Tg raises Cai to values greater and for longer than calcium requiring agonists only the latter affected transepithelial transport processes. It is shown that Tg neither affects transepithelial sodium transport nor chloride conductance, both of which increase in response to lysylbradykinin or ATP. It is concluded that spatio-temporal patterns of Cai increase after Tg and other agonists are different.
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PMID:Failure of thapsigargin to alter ion transport in human sweat gland epithelia while intracellular Ca2+ concentration is raised. 132 17

In the preceding paper it was shown that an isoform of serum albumin (ASA; active serum albumin) causes a rapid retraction of neurites and increases intracellular content of Ins1,4,5P3 in PC12 cells. Here we examined whether ASA's effects in nerve growth factor-differentiated PC12 cells were mediated through the Ins1,4,5P3/Ca2+ second messenger pathway by monitoring intracellular Ca2+ (Ca2+i) with Fura2. It was found that ASA caused a dose-dependent increase in Ca2+i. In Ca(2+)-free medium, the increase in Ca2+i elicited by ASA was smaller, but the rise in Ins1,4,5P3 content was not appreciably changed. The small Ca2+i increase seen in Ca(2+)-free medium was probably due to the release of Ca2+ from Ins1,4,5P3-sensitive intracellular stores. In Ca(2+)-containing medium the Ca2+ transient induced by ASA was not affected by organic Ca2+ channel blockers, but decreased when Co2+, Mn2+ or Zn2+ were present in the extracellular medium. The effect of other ligands, such as carbachol and bradykinin, whose receptors are coupled to the phosphoinositide system was also investigated. Carbachol at concentrations from 2 to 200 microM, and bradykinin at a concentration of 2 microM did not cause neurite retraction, whereas 200 microM bradykinin caused an approximately 40% decrease in neurite length. Thapsigargin, a Ca(2+)-ATPase inhibitor, caused a sustained elevation of Ca2+i and retraction of neurites, whereas depolarization of the cells by high K+ gave only a transient elevation of Ca2+i, and no neurite retraction. Therefore, a sustained elevation in Ca2+i might be a sufficient trigger to induce neurite retraction in differentiated PC12 cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The effect of active serum albumin on PC12 cells: II. Intracellular Ca2+ transients and their role in neurite retraction. 132 93

Thapsigargin (Tg) effects on Ca2+ handling in the intact human platelet were studied using Quin2 and chlorotetracycline to measure free cytoplasmic and dense tubular (DT) Ca2+ concentrations ([Ca2+]cyt and [Ca2+]dt, respectively). Tg inhibits Ca2+ uptake by the DT Ca(2+)-ATPase pumps, but incompletely, lowering the Vm to 32% of control (IC50,Tg = 0.18 +/- 0.10 microM). The kinetics of loss of DT Ca2+, transient increases in [Ca2+]cyt, and lowered steady-state [Ca2+]dt after Tg addition are all explained by pump inhibition, with no effect on the rate constant of Ca2+ leakage across the DT membrane (kleak,DT = 1.14 min-1). Tg lowers by 30% the Vm of the Ca2+ extrusion pump located in the plasma membrane (PM), as shown by a Quin2-based method measuring active Ca2+ extrusion (Johansson, J. S., and Haynes, D. H. (1988) J. Membr. Biol. 104, 147-163). This effect (IC50,Tg = 0.45 +/- 0.06 microM), together with a 24 +/- 16% increase in kleak,PM,Ca (to 3 x 10(-4) min-1), accounts for a Tg-dependent sustained elevation [Ca2+]cyt (to 708 +/- 78 nM) which is independent of DT Ca2+ status or history. Thrombin and Tg release 30 and 70% (respectively) of the DT Ca2+ available at any instant, independent of order of challenge, consistent with a single class of DT with respect to these agents.
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PMID:Actions of thapsigargin on the Ca(2+)-handling systems of the human platelet. Incomplete inhibition of the dense tubular Ca2+ uptake, partial inhibition of the Ca2+ extrusion pump, increase in plasma membrane Ca2+ permeability, and consequent elevation of resting cytoplasmic Ca2+. 133 75

The regulation of the intracellular free Mg2+ concentration ([Mg2+]i) was monitored in rat sublingual mucous acini using dual wavelength microfluorometry of the Mg(2+)-sensitive dye mag-fura-2. Acini attached to coverslips and superfused continuously with a Mg(2+)-containing medium (0.8 mM) have a steady-state [Mg2+]i of 0.35 +/- 0.01 mM. Adjusting the extracellular Mg2+ concentration to 0 and 10 mM or removing extracellular Na+ did not alter the resting [Mg2+]i. Stimulation with the Ca(2+)-mobilizing, muscarinic agonist, carbachol, induced a sustained increase in [Mg2+]i (approximately 50%; t1/2 < 20 s; Kd approximately 1.5 microM), the magnitude and the duration of which were unchanged in Mg(2+)-depleted medium indicating that the rise in [Mg2+]i was generated by Mg2+ release from an intracellular Mg2+ pool. Forskolin, which increases the intracellular cAMP content, produced a small, transient increase in the [Mg2+]i (< 10%). Muscarinic stimulation in a Ca(2+)-free medium blunted the initial increase in [Mg2+]i by approximately 50%, whereas the sustained increase in [Mg2+]i was lost. When the muscarinic-induced increase in [Ca2+]i was blocked by 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate, an inhibitor of the agonist-sensitive intracellular Ca2+ release pathway, both the initial and the sustained phases of the increase in [Mg2+]i were virtually eliminated. Thapsigargin and 2,5-di-(terbutyl)-1,4-benzohydroquinone, which increase [Ca2+]i by inhibiting microsomal Ca(2+)-ATPase, caused a dramatic increase in [Mg2+]i. Stimulation in a Na(+)-free medium or in the presence of bumetanide, an inhibitor of Na+/K+/2Cl- cotransport, blunted the agonist-induced rise in [Mg2+]i (approximately 50%), whereas ouabain, a Na+,K(+)-ATPase inhibitor, had no significant effect. FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), a mitochondrial uncoupler, mobilized an intracellular Mg2+ pool as well. The carbachol-induced increase in [Mg2+]i was markedly inhibited by FCCP (approximately 80%), suggesting that the same pool(s) of Mg2+ were primarily involved. The above results provide strong evidence that Ca(2+)-mobilizing agonists increase cytoplasmic free [Mg2+] by releasing an intracellular pool of Mg2+ that is associated with a rise in the [Na+]i.
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PMID:Secretagogue-induced mobilization of an intracellular Mg2+ pool in rat sublingual mucous acini. 138 8

The free Ca2+ concentrations in the nucleus ([Ca2+]n) and cytoplasm ([Ca2+]c) of cultured smooth muscle cells were estimated using the fluorescent dye indo-1 and the ACAS 570 confocal laser microscope. In resting DDT1MF2 smooth muscle cells [Ca2+]n was found to be lower than [Ca2+]c. Both values increased transiently in response to histamine (100 microM), but during this stimulation [Ca2+]n exceeded [Ca2+]c. Maximal increase of [Ca2+]n was observed in the center of the nucleus, and a maximal increase of [Ca2+]c was observed in the immediate vicinity of the plasma membrane. A similar response was obtained with other agonists, such as carbachol or ATP. Comparable results with ATP were obtained in cultured aorta cells. The differential rise of [Ca2+]n over [Ca2+]c in DDT1MF2 cells did not occur during either spontaneous release of Ca2+ or Ca2+ release induced by caffeine (7.5 mM). The differential rise during histamine stimulation was abolished by the presence of the intercalating substance ethidium bromide. Thapsigargin, a presumed specific inhibitor of the endoplasmic reticulum Ca(2+)-Mg(2+)-adenosine-triphosphatase, abolished the Ca2+ gradient between nucleus and cytosol at rest. During subsequent histamine stimulation the Ca2+ increase was largely blocked in both compartments and attained similar levels. We propose that the lower value of [Ca2+]n at rest is dependent on an active Ca2+ extrusion system. The differential rise of [Ca2+]n over [Ca2+]c during agonist stimulation can be explained by an influx of Ca2+ from perinuclear stores and/or by a release of intranuclear Ca2+ possibly mediated by a process dependent on the inositol lipid metabolism.
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PMID:Differences in regulation between nuclear and cytoplasmic Ca2+ in cultured smooth muscle cells. 138 89

Continuous superfusion of rat glioma cells with medium containing bradykinin (from 0.2 nM) induced a transient hyperpolarization followed by regular hyperpolarizing oscillations of the membrane potential. Similar repetitive hyperpolarizing oscillations were caused by extracellularly applied bradykinin or muscarine or by intracellularly injected GTP-gamma-S. The frequency of the oscillations was 1 per minute at bradykinin concentrations ranging from 0.2 nM to 2 microM, but the amplitude and duration increased with rising peptide concentration. The muscarine-induced oscillations were blocked by atropine. In the presence of extracellular Ca2+, the substances thapsigargin, 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), and ionomycin reversibly suppressed the bradykinin-induced oscillations. Thapsigargin and tBuBHA, which are known to block the Ca2+ ATPase of endoplasmic reticulum, caused a transient rise in cytosolic Ca2+ activity, monitored with Fura-2, in suspensions of rat glioma cells or of mouse neuroblastoma-rat glioma hybrid cells. After a transient Ca2+ rise caused by thapsigargin, tBuBHQ, or ionomycin, the Ca2+ response to bradykinin which is known to be due to release of Ca2+ from internal stores was suppressed. This indicates that thapsigargin and tBuBHQ deplete internal Ca2+ stores as already seen previously for ionomycin. Thus, the inhibition of the membrane potential oscillations by thapsigargin, tBuBHQ, and ionomycin indicates that the oscillations are associated with activation of InsP3-sensitive Ca2+ stores. In some cells composite oscillation patterns which consisted of two independent oscillations with different amplitudes that overlapped additively were seen. We discuss that this pattern and the concentration dependency of the oscillations could be due to "quantal" Ca2+ release from stores with different inositol 1,4,5-triphosphate sensitivities. Subsidence of the oscillations after omission of extracellular Ca2+ seems to be due to a lack of replenishment of the intracellular stores with Ca2+, which comes from the extracellular compartment.
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PMID:Bradykinin and muscarine induce Ca(2+)-dependent oscillations of membrane potential in rat glioma cells indicating a rhythmic Ca2+ release from internal stores: thapsigargin and 2,5-di(tert-butyl)-1, 4-benzohydroquinone deplete InsP3-sensitive Ca2+ stores in glioma and in neuroblastoma-glioma hybrid cells. 139 96

Cytosolic free Ca2+ rises in pancreatic beta-cells in response to glucose stimulation and is part of the coupling to insulin secretion. This study evaluates a possible role for cytosolic long chain acyl-CoA esters in modulating Ca2+ handling by clonal beta-cells (HIT). Intact cells incubated with 20 microM free palmitic acid exhibited a 40% decrease in basal cytosolic free Ca2+. In contrast, acyl-CoA esters, up to a chain length of 16, but not the corresponding fatty acids, significantly lowered the Ca2+ set point maintained by cells permeabilized with saponin. The maximum response to the various acyl-CoA esters increased with increasing chain length, with no differences in the half-maximally effective concentration of 0.5 microM. Long chain acyl-CoA esters caused a 40-50% increase in 45Ca2+ influx into a non-mitochondrial pool in the permeabilized HIT cells, consistent with a stimulatory effect on the endoplasmic reticulum Ca(2+)-ATPase activity, but did not affect inositol 1,4,5-trisphosphate-induced Ca(2+)-efflux. Thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+)-ATPase activity, blocked the decrease in the Ca2+ set point caused by acyl-CoA esters. The ability of acyl-CoA esters to lower the Ca2+ set point depended on the ATP/ADP ratio (or free ADP); the Ca2+ set point was lowered by 36 +/- 3.6% at an ATP/ADP ratio of 90 and by 14 +/- 1.9% at an ATP/ADP ratio of 7. Depletion of cellular protein kinase C did not prevent the acyl-CoA-induced lowering of the Ca2+ set point. These findings suggest that the increases in long chain acyl-CoA esters may play a role in restoring cytosolic free Ca2+ through activation of Ca(2+)-ATPases.
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PMID:Acyl-CoA esters modulate intracellular Ca2+ handling by permeabilized clonal pancreatic beta-cells. 140 Mar

I report here that thapsigargin, an inhibitor of Ca(2+)-ATPase activities in internal Ca2+ stores, induces meiotic maturation in prophase I-arrested surf clam (Spisula solidissima) oocytes. The half-maximal dose for triggering germinal vesicle breakdown (GVBD) is 120 nM. Thapsigargin-induced GVBD is followed by all normal subsequent steps of meiotic maturation including extrusions of first and second polar bodies, with almost normal timing as compared with K(+)-induced activation. Thapsigargin-induced GVBD requires the presence of external Ca2+ at a half-maximal concentration of 0.6 mM. In normal sea water, thapsigargin-induced activation is accompanied by a slightly increased 45Ca2+ uptake by the oocytes and by an intracellular pH rise of 0.3 U. These results show that thapsigargin-sensitive Ca2+ pools regulating Ca2+ fluxes exist in surf clam oocytes, and they also further establish that Ca2+ ions are the major initial trigger for meiosis resumption in this species.
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PMID:Thapsigargin induces meiotic maturation in surf clam oocytes. 144 7

Thapsigargin is a potent inhibitor of the microsomal calcium-ATPase. We hypothesized that thapsigargin-induced liberation of calcium from sequestered intracellular sites could result in irreversible brain injury. To assess neurotoxicity, intracerebral injections of 0.1-100 nmol thapsigargin were performed in 7-day-old rats, and the severity of injury was assessed 5 days later by microscopy and measurements of lesioned areas. We found that thapsigargin elicited dose-dependent tissue injury in perinatal rodent brain.
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PMID:The microsomal calcium-ATPase inhibitor thapsigargin is a neurotoxin in perinatal rodent brain. 146 12


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