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)

Regulation of cytosolic Ca2+ and cytosolic Na+ is critical for lymphocyte cation homeostasis and function. To examine the influence of cytosolic Na+ on Ca2+ regulation in human peripheral blood lymphocytes, Ca2+ entry and cytosolic Ca2+ (measured with fura-2) were monitored in cells in which cytosolic Na+ was increased and/or the Na+ gradient was decreased by reduction of external Na+ concentration. Ouabain-treated cells (0.1 mM for 30 min at 37 degrees C), suspended in Na(+)-free medium, showed a 30-65% increase in Ca2+ uptake compared to cells in 140 mM Na+ medium. Enhanced Ca2+ influx was entirely dependent on ouabain pretreatment and reversal of the Na+ gradient. Na pump inhibition or Na ionophore addition and subsequent exposure to Na(+)-free medium resulted in a sustained elevation of cytosolic Ca2+. As preincubation of cells in Ca(2+)-free medium further enhanced the ouabain-dependent increase in cytosolic Ca2+, the effects of the microsomal Ca(2+)-ATPase inhibitor thapsigargin on Ca2+ influx and cytosolic Ca2+ were studied. Thapsigargin stimulated Ca2+ entry following ouabain pretreatment and reversal of the Na+ gradient; the effects of thapsigargin were retained in the presence of LaCl3, a potent inhibitor of store-dependent calcium influx pathways. These results show lymphocytes demonstrate Na+/Ca2+ exchange activity and suggest the Na+/Ca2+ exchanger modulates cytosolic Ca2+ following intracellular Ca2+ store depletion.
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PMID:Na+/Ca2+ exchange-mediated calcium entry in human lymphocytes. 796 46

The activation of muscarinic receptors in N1E-115 neuroblastoma cells elicits a voltage-independent calcium current. The current turns on slowly, reaches its maximum value approximately 45 s after applying the agonist, is sustained as long as agonist is present, and recovers by one half in approximately 10 s after washing the agonist away. The current density is 0.11 +/- 0.08 pA/pF (mean +/- SD; n = 12). It is absent in zero-Ca++ saline and reduced by Mn++ and Ba++. The I(V) curve characterizing the current has an extrapolated reversal potential > +40 mV. The calcium current is observed in cells heavily loaded with BAPTA indicating that the calcium entry pathway is not directly gated by calcium. In fura-2 experiments, we find that muscarinic activation causes an elevation of intracellular Ca++ that is due to both intracellular calcium release and calcium influx. The component of the signal that requires external Ca++ has the same time course as the receptor operated calcium current. Calcium influx measured in this way elevates (Ca++)i by 89 +/- 41 nM (n = 7). Thapsigargin, an inhibitor of Ca++/ATPase associated with the endoplasmic reticulum (ER), activates a calcium current with similar properties. The current density is 0.22 +/- 0.20 pA/pF (n = 6). Thapsigargin activated current is reduced by Mn++ and Ba++ and increased by elevated external Ca++. Calcium influx activated by thapsigargin elevates (Ca++)i by 82 +/- 35 nM. The Ca++ currents due to agonist and due to thapsigargin do not sum, indicating that these procedures activate the same process. Carbachol and thapsigargin both cause calcium release from internal stores and the calcium current bears strong similarity to calcium-release-activated calcium currents in nonexcitable cells (Hoth, M., and R. Penner. 1993. Journal of Physiology. 465:359-386; Zweifach, A., and R. S. Lewis, 1993. Proceedings of the National Academy of Sciences, USA. 90:6295-6299).
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PMID:Calcium current activated by muscarinic receptors and thapsigargin in neuronal cells. 796 92

We examined the effect of the depletion of intracellular Ca2+ stores on Ca2+ influx in rat glomerulosa cells. Depletion of intracellular Ca2+ stores was achieved by inhibiting sarco/endoplasmic reticulumtype Ca(2+)-ATPase with thapsigargin or 2,5,di-(t-butyl)-1,4-benzohydroquinone (t-BHQ). Both inhibitors induced a sustained rise in cytoplasmic Ca2+ concentration. The initial rise was observed also in Ca(2+)-free medium, while the sustained phase disappeared, indicating that the latter requires Ca2+ influx. In Ca(2+)-free medium, the readdition of Ca2+ induced a steeper and higher rise in intracellular Ca2+ concentration in thapsigargin-treated cells than in controls, supporting the role of Ca2+ influx. In normal medium, the addition of Cd2+ (80 microM) evoked an immediate inhibition of the sustained phase of thapsigargin response. The response to thapsigargin was insensitive to nifedipine. Thapsigargin failed to enhance Mn2+ quenching of fura 2. Our results provide evidence for the existence of capacitative Ca2+ influx in rat glomerulosa cells and indicate that dihydropyridine-sensitive Ca2+ channels do not participate in capacitative Ca2+ entry. High concentrations of thapsigargin and t-BHQ, similar to the reported effects of angiotensin II and vasopressin, inhibited K(+)-induced Ca2+ signals. These effects appear, however, to be independent of the depletion of internal Ca2+ stores.
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PMID:Capacitative Ca2+ influx in adrenal glomerulosa cells: possible role in angiotensin II response. 797 88

On reconstitution of the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum into bilayers of dimyristoleoylphosphatidylcholine [di(C14:1)PC] or dinervonylphosphatidylcholine [di(C24:1)PC] the stoichiometry of Ca2+ binding changes from the usual two Ca2+ ions bound per ATPase molecule to one Ca2+ ion bound per ATPase molecule. For the ATPase in di(C24:1)PC, removal of Ca2+ from the Ca(2+)-bound ATPase results in a decrease in tryptophan fluorescence intensity, as observed for the ATPase in dioleoylphosphatidylcholine [di(C18:1)PC]. For the ATPase in di(C14:1)PC removal of Ca2+ results in no change in tryptophan fluorescence intensity. In the presence of Mg2+, removal of Ca2+ from the ATPase in di(C18:1)PC or di(C24:1)PC results in a decrease in tryptophan fluorescence intensity, but for the ATPase in di(C14:1)PC this results in an increase in intensity. Fluorescence of the ATPase labelled with 4-nitrobenzo-2-oxa-1,3-diazole (NBD) is the same for the ATPase in di(C18:1)PC or di(C24:1)PC, but is markedly greater in di(C14:1)PC, consistent with a 4-fold increase in the E1/E2 equilibrium constant. Addition of Mg2+ to NBD-labelled ATPase in di(C18:1) PC or di(C24:1)PC results in an increase in NBD fluorescence, attributed to stronger binding of Mg2+ to the E1 than to the E2 conformation; addition of Mg2+ had no effect on the fluorescence of the NBD-labelled ATPase in di(C14:1)PC. In the absence of Ca2+, Mg2+ increased the tryptophan fluorescence of the ATPase in di(C14:1)PC, di(C18:3)PC or di(C24:1)PC, with the same binding-constant for Mg2+ in all three lipids. Addition of Mg2+ to the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin resulted in a decrease in fluorescence in di(C18:1)PC or di(C24:1)PC but had no effect in di(C14:1)PC. These effects are interpreted in terms of binding of Ca2+ at a single outer Ca2+ binding-site on the ATPase in di(C14:1)PC and di(C24:1)PC, in a conformation in which the inner site is occluded [in di(C14:1)PC] or modified in its affinity for Ca2+ [in di(C24:1)PC]. Thapsigargin binds to the ATPase, reducing its affinity for Ca2+ both in di(C14:1)PC and di(C24:1)PC.
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PMID:Characterization of the single Ca(2+)-binding site on the Ca(2+)-ATPase reconstituted with short- or long-chain phosphatidylcholines. 799 94

The endomembrane Ca(2+)-ATPase inhibitor, thapsigargin, was used to deplete the intracellular Ca2+ stores of fura-2-loaded human platelets. In control cells, thapsigargin evoked a rise in cytosolic [Ca2+] and a substantial increase in protein tyrosine phosphorylation. Thapsigargin also evoked an increase in tyrosine phosphorylation in cells co-loaded with fura-2 and the Ca2+ chelator dimethyl BAPTA, such that the rise in cytosolic [Ca2+] was abolished. These data support the existence of a tyrosine phosphatase regulated by the Ca2+ content of the intracellular store, a requirement of the putative model for reciprocal control of Ca2+ entry by cytosolic and store [Ca2+] via protein tyrosine phosphorylation.
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PMID:Calcium store depletion in dimethyl BAPTA-loaded human platelets increases protein tyrosine phosphorylation in the absence of a rise in cytosolic calcium. 800 12

Furazolidone is a nitrofuran antibiotic that causes dilated cardiomyopathy in turkeys and chicks and serves as an important model of human dilated cardiomyopathy. The mechanism by which furazolidone produces cardiac injury remains unknown. We investigated the hypothesis that furazolidone alters Ca2+ homeostasis in cardiac muscle cells. Myocytes harvested from 7-day-old chick embryos were treated with furazolidone (0.02, 0.1, and 1 mM) for 24-52 h and then coloaded with seminaphthorhodafluor-1 (SNARF 1) and fura 2 to measure simultaneously intracellular pH (pHi) and intracellular Ca2+ concentration ([Ca2+]i), respectively. Furazolidone did not affect steady-state [Ca2+]i levels in cardiac myocytes. Na+ removal was associated with a rapid increase in [Ca2+]i due to the Na+/Ca2+ exchanger, which was similar in control and furazolidone-treated cells. The rate of [Ca2+]i recovery after Na+ removal was significantly increased in the furazolidone-treated cells compared with controls. In most cells, recovery from Ca2+ load is accomplished by the activity of Ca(2+)-adenosinetriphosphatases (ATPases). Thapsigargin, inhibitor of sarcoplasmic reticulum Ca(2+)-ATPase, prevented the furazolidone-induced changes. These results demonstrate that furazolidone increases the activity of thapsigargin-sensitive Ca(2+)-ATPase without affecting Na+/Ca2+ exchange. These data enhance our understanding of the mechanism of furazolidone-induced injury in cardiac myocytes and may be useful in determining mechanisms of injury in dilated cardiomyopathy.
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PMID:Furazolidone increases thapsigargin-sensitive Ca(2+)-ATPase in chick cardiac myocytes. 806 29

In many cell types, emptying of intracellular Ca2+ stores after application of inhibitors of the intracellular Ca(2+)-ATPase (e.g. thapsigargin) is astonishingly rapid. It was the aim of this study to elucidate the underlying mechanism. We first compared thapsigargin-induced emptying of intracellular Ca2+ stores in intact and homogenized HL-60 granulocytes. Thapsigargin-induced Ca2+ release was rapid in intact cells (33.9 +/- 4.9% of store content/min), but it was slow in permeabilized or homogenized cells (7.7 +/- 3.9 and 12 +/- 3.8% of store content/min respectively). To study whether the Ins(1,4,5)P3 receptor might be involved in thapsigargin-induced Ca2+ release, we tested the effect of heparin, a competitive Ins(1,4,5)P3 antagonist. In homogenized and permeabilized preparations, heparin did not interfere with thapsigargin-induced Ca2+ release. In contrast, when introduced into intact cells by an endocytosis/osmotic-shock procedure, heparin, but not the inactive de-N-sulphated heparin, decreased the rate of Ca2+ release by approx. 70%. Heparin inhibited Ca2+ release in response to the Ins(1,4,5)P3-generating receptor agonist N-formylmethionyl-leucyl-phenylalanine (f-MLP) (50 nM) and to thapsigargin (50 nM) at comparable concentrations. Heparin inhibition was competitive for f-MLP-induced, but not for thapsigargin-induced, Ca2+ release. In permeabilized cells, the addition of low Ins(1,4,5)P3 concentrations before thapsigargin increased the rate of thapsigargin-induced Ca2+ release 4-fold. Taken together, our results suggest that the rapid Ca(2+)-ATPase-inhibitor-induced Ca2+ release is due to a partial activation of the Ins(1,4,5)P3 receptor in resting cells. This implies Ca2+ cycling across the membrane of Ins(1,4,5)P3-sensitive Ca2+ stores in resting cells.
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PMID:Rapid heparin-sensitive Ca2+ release following Ca(2+)-ATPase inhibition in intact HL-60 granulocytes. Evidence for Ins(1,4,5)P3-dependent Ca2+ cycling across the membrane of Ca2+ stores. 806 1

Thapsigargin induced endothelium-dependent relaxation and cGMP production in rat thoracic aorta, and these effects were inhibited by nitric oxide (NO) pathway inhibitors, a calmodulin inhibitor and removal of Ca2+, suggesting that NO is involved in the thapsigargin-induced relaxation. Thapsigargin may deplete Ca2+ stores in the endothelial cells by inhibiting the Ca(2+)-ATPase, a Ca2+ pump, which in turn triggers influx of extracellular Ca2+, leading to activation of constitutive NO synthase and resultant NO generation. The NO thus formed may activate soluble guanylate cyclase to produce cGMP in the vascular smooth muscle.
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PMID:Thapsigargin, a Ca(2+)-ATPase inhibitor, relaxes rat aorta via nitric oxide formation. 811 11

The dynamics of intracellular Ca2+ signalling in single melanotrope cells of the pituitary gland of the amphibian Xenopus laevis have been studied by means of a digital imaging technique using the fluorescent dye Fura-2. When placed in vitro, the majority of the cells (77%) displayed spontaneous oscillatory changes in the free cytosolic Ca2+ concentration with a frequency of 1 +/- 0.25 (SD) min-1. The oscillations rapidly stopped when extracellular Ca2+ was reduced to nanomolar concentrations, revealing their complete dependence on Ca2+ influx. The fact that the Ca2+ oscillations were blocked by 1 microM omega-conotoxin, but not by nifedipine, at concentrations up to 50 microM, indicated that Ca2+ entered the cell via N-type rather than L-type voltage operated Ca2+ channels. Thapsigargin, a putative inhibitor of intracellular Ca(2+)-ATPase activity, elevated the baseline Ca2+ concentration but had no effect on the occurrence of the spontaneous oscillations. This suggests that intracellular Ca2+ pools are not involved in the mechanism underlying spontaneous Ca2+ oscillations. This is the first report showing spontaneous Ca2+ oscillations mediated by N-type Ca2+ channels in melanotrope cells.
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PMID:Spontaneous calcium oscillations in Xenopus laevis melanotrope cells are mediated by omega-conotoxin sensitive calcium channels. 814 4

Thapsigargin, an inhibitor of several isoforms of the Ca(2+)-ATPase protein, has been used in many cell preparations to induce an increase in cytosolic Ca2+ concentration purportedly by inhibition of the catalytic cycle. We report in this paper, that thapsigargin induces rapid Ca2+ release from sarcoplasmic reticulum vesicles at concentrations higher than those required to inhibit ATPase activity. Thapsigargin also induces a similar concentration-dependent release in Ca(2+)-loaded asolectin liposomes devoid of any protein. These data suggest that Ca2+ release induced by micromolar concentrations of thapsigargin is due to an ionophoric effect on the lipid membrane.
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PMID:Thapsigargin-induced Ca2+ release from sarcoplasmic reticulum and asolectin vesicles. 814 18


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