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

---

Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Thapsigargin (TG), a plant sesquiterpene lactone extract, interacts tightly with the sarcoplasmic reticulum (SR) Ca2+ transport ATPase yielding a 1:1 stoichiometric complex. In addition to inhibiting steady state enzyme activity, TG can be shown to inhibit two individual partial reactions of the ATPase cycle (i.e. Ca2+ binding in the absence of ATP and enzyme phosphorylation by Pi in the absence of Ca2+) even when these reactions are studied separately without interdependence. As the two partial reactions occur at domains relatively distant from each other in the protein structure, it is apparent that the TG induced perturbation involves the entire enzyme. The rate of TG interaction with the ATPase, as estimated by the onset of functional inhibition and by the development of an intrinsic fluorescence signal, is relatively low in the presence of Ca2+. The interaction is much faster when Ca2+ is removed from the medium by the addition of EGTA or is dissociated from the enzyme by utilization of ATP. When the TG interaction with the ATPase is studied in the presence of Ca2+ as a function of temperature (15-35 degrees C) and pH (6.0-8.0), two distinct kinetic components are observed: a fast component which is prevalent at high temperature and low pH, and a slow component which is prevalent at low temperature and high pH. This pattern suggests that the enzyme resides in two states, whose relatively slow equilibration is temperature- and pH-dependent. As only one state is reactive to TG, the enzyme population residing in this state reacts immediately with TG. On the other hand, the enzyme population residing in the alternate state must undergo slow conversion to the reactive state before being affected by TG. It can be also demonstrated that in the presence of Ca2+ TG shifts the ATPase from a refractory state to a state which is able to form bidimensional crystalline arrays stabilized by decavanadate. It is concluded that TG reacts specifically with the ATPase conformation which is prevalent in the absence of Ca2+, thereby forming a catalytically inactive dead-end complex.
...
PMID:A conformational mechanism for formation of a dead-end complex by the sarcoplasmic reticulum ATPase with thapsigargin. 153 Sep 36

Thapsigargin, which acts by inhibition of a Ca(++)-ATPase on the dense tubule system in platelets, is a pharmacological tool to study the effects of increases in intracellular Ca++. Secondary consequences of thapsigargin treatment in platelets include extensive thromboxane B2 formation (493 +/- 106 ng/10(8) platelets) and [3H]5-hydroxytryptamine secretion (80.7 +/- 8.0%). Inhibition of cyclooxygenase by ibuprofen prevents thromboxane B2 formation (0.1 +/- 0.04 ng/10(8) platelets) and dense tubule secretion (6.5 +/- 3.8%). Aggregation in response to thapsigargin is rapid and maximal, but the rate and extent of aggregation are lowered by ibuprofen or aspirin. Mobilization of intracellular Ca++ is also significantly attenuated when eicosanoid formation is prevented, indicating the dependence of thapsigargin actions on endogenous lipid mediator formation. These studies also support the idea that formation of endogenous thromboxane A2/prostaglandin H2 is self-amplifying; thromboxane receptor antagonists inhibit endogenous thromboxane B2 formation, indicating that Ca(++)-dependent activation of phospholipase A2 is only partially responsible for eicosanoid production. Our data indicate the importance of distinguishing secondary effects of thapsigargin, especially because it may influence eicosanoid formation.
...
PMID:Indirect actions of thapsigargin on human platelets: activation of eicosanoid biosynthesis and cellular signaling pathways. 153 33

The recently developed nystatin modification of the patch clamp technique allows stable whole-cell recordings without affecting the intracellular Ca2+ buffering capacity and thereby may provide a means to indirectly monitor spontaneous changes in the intracellular Ca2+ concentrations. To test this hypothesis, we applied the nystatin method to the well-characterized ROS 17/2.8 osteoblast-like cell system, where rises of the intracellular Ca2+ are known to cause transient hyperpolarizations via activation of Ca2+ -dependent K+ channels. Additionally to minor fluctuations (10-20 mV) around a mean potential of -42.1 +/- 4.2 mV, we observed spontaneously occurring, transient hyperpolarizations to membrane potentials as negative as -80 mV. These transient hyperpolarizations were not eliminated by Ca2+ entry blockers but abolished by intracellular infusion of 10 mM EGTA. Thapsigargin, a specific inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, hyperpolarized the cells close to the K+ reversal potential. Moreover, voltage-clamp studies revealed an intermittendly activating Ca2+-dependent K+ conductance. These results strongly suggest that the nystatin method is particularly suitable to study Ca(2+)-dependent channels and thereby spontaneous changes in the intracellular Ca2+.
...
PMID:Transient membrane hyperpolarizations due to spontaneous fluctuations of the cytosolic Ca2+ in osteoblast-like cells. 153 49

We have investigated the role of the sarcoplasmic reticulum Ca2+ pool in regulating Ca2+ entry in vascular smooth muscle cells using a receptor-independent means of mobilizing the intracellular Ca2+ pool. Thapsigargin (TG) has been shown to inhibit the endoplasmic reticulum Ca(2+)-ATPase, mobilize intracellular Ca2+, and activate Ca2+ entry in nonmuscle tissues. When smooth muscle cells were treated with 0.2 microM TG, cytosolic Ca2+ concentrations rose gradually over 8 min to a peak value of 365 +/- 18 nM. Cytosolic Ca2+ remained elevated for at least 20 min and was supported by continued entry of extracellular Ca2+. TG also stimulated entry of Mn2+ and 45Ca2+ from outside the cell. Importantly, TG-induced Ca2+ entry and Mn2+ entry were found to occur through mechanisms that were independent of L-type Ca2+ channel activation because influx was not inhibited by concentrations of nicardipine that were found to block either endothelin- or 100 mM extracellular K(+)-induced cation influx. The mechanism through which TG activates cation entry appears to involve mobilization of the inositol 1,4,5-trisphosphate-responsive intracellular Ca2+ pool. In permeabilized cells, TG prevented ATP-stimulated Ca2+ uptake into the sarcoplasmic reticulum and slowly released sequestered Ca2+. The Ca2+ pool involved was responsive to inositol 1,4,5-trisphosphate. However, TG did not initiate the formation of inositol polyphosphates. Thus TG mobilizes the sarcoplasmic reticulum Ca2+ pool and activates Ca2+ entry through a nicardipine-insensitive Ca2+ channel in vascular smooth muscle. The mechanism is independent of inositol polyphosphate formation.
...
PMID:Thapsigargin stimulates Ca2+ entry in vascular smooth muscle cells: nicardipine-sensitive and -insensitive pathways. 153 1

Sustained GnRH-stimulated LH release requires extracellular Ca2+, but GnRH transiently increases LH release in Ca(2+)-free medium. Here we have tested the dependence of the transient effect on intracellular Ca2+ pools. In superfused pituitary cells three Ca(2+)-mobilizing stimuli (GnRH, A23187, and endothelin-1) all caused sustained increases in LH release in normal medium (plateau responses), but only transient increases in Ca(2+)-free medium (spike responses). In Ca(2+)-free medium, GnRH (10(-10) or 10(-9) M) increased LH release transiently and desensitized the cells to the LH-releasing effect of subsequent stimulation with 10(-7) M GnRH. This desensitization was reversed by brief exposure to Ca(2+)-containing medium between the two GnRH stimulation periods. Heterologous desensitization between GnRH and A23187 and between GnRH and endothelin-1 also occurred in Ca(2+)-free medium. Thapsigargin, which inhibits the endoplasmic reticulum Ca(2+)-ATPase and thereby elevates cytosolic Ca2+, stimulated LH release (EC50, approximately 20 microM) in static culture, an effect which, unlike those of GnRH and A23187, was not markedly reduced in Ca(2+)-free medium. Low doses of thapsigargin, which had no effect on LH release alone, inhibited both sustained GnRH-stimulated LH release from static cultures in normal medium and transient GnRH-stimulated LH release from cells superfused in Ca(2+)-free medium. These data suggest that the spike phase of GnRH-stimulated LH release is not only associated with but is also dependent upon the mobilization of a GnRH- and thapsigargin-sensitive intracellular Ca2+ pool and that the Ca2+ pool mediating this GnRH effect is identical to or substantially interchangeable with A23187- and endothelin-1-mobilizable intracellular Ca2+ pools. Inhibition of sustained GnRH-stimulated LH release by thapsigargin also suggests the involvement of an intracellular Ca2+ pool in this phase of GnRH action.
...
PMID:Dependence of gonadotropin-releasing hormone-stimulated luteinizing hormone release upon intracellular Ca2+ pools is revealed by desensitization and thapsigargin blockade. 153 42

Bovine endothelial cell monolayers grown to confluence and stimulated with bradykinin responded with periodic fluctuations in intracellular Ca2+ concentration ([Ca2+]i) when exposed to K(+)-free Hepes-buffered saline. The fluctuations in [Ca2+]i measured with fura-2 were synchronized among the population of cells observed and were sensitive to extracellular Ca2+ concentration ([Ca2+]o). Thapsigargin, which inhibits the endoplasmic reticular Ca2(+)-ATPase, did not inhibit the [Ca2+]i oscillations. Removal of extracellular Ca2+ or inhibition of Ca2+ entry by using La3+ or 1-(beta- [3-(4-methoxyphenyl)proproxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SKF 96365) abolished the [Ca2+]i oscillations in endothelial cell monolayers. The fluctuations in [Ca2+]i were therefore dependent on Ca2+ influx rather than Ca2+ mobilization from intracellular stores. Simultaneous measurements of membrane potential (Em) using the potential-sensitive bisoxonol dye bis(1,3-dibutylbarbituric acid)trimethine oxonol [Di-BAC4(3)] and [Ca2+]i using fura-2 showed that Em oscillated at the same frequency as the fluctuations in [Ca2+]i. The peak depolarization signal coincided with the maximum rate of increase in the [Ca2+]i signal. Oscillations in the Em signal were inhibited by removal of Ca2+ or by addition of 1 mM Ni2+ to the external solution. Taken together, these observations suggest that the change in Em is the consequence of oscillatory changes in a membrane conductance that also allows Ca2+ to enter the cell. Oscillations in the DiBAC4(3) signal may reflect a rhythmic entry of Ca2+ through nonselective cation channels.
...
PMID:Calcium entry-dependent oscillations of cytoplasmic calcium concentration in cultured endothelial cell monolayers. 154 61

In this report, we describe a Jurkat cell variant, termed JCT8, the selection of which is based upon its resistance to cell-growth inhibition mediated by the holotoxin of Vibrio cholerae, cholera toxin (CT). JCT8 cells exhibit normal cAMP production in response to various cAMP inducers, including CT, together with conserved ADP ribosylation in vitro of G-protein Gs alpha by the A subunit of the toxin. However, after a 4-h pretreatment with CT, JCT8 cells have a conserved expression of cell-surface CD3 molecules. These effects are in contrast to those elicited by the toxin in long term PGE2-desensitized Jurkat cells, which remain as sensitive as the wild type to the inhibitory action of CT on cell growth and CD3 cell-surface expression, despite poor responsiveness to CT with regard to cAMP production. In JCT8 cells, Ca2+ mobilization induced via the CD3/TCR is maintained after CT treatment contrasting with its complete suppression in the wild-type and in the PGE2-desensitized cells. However, as in the other cell types, CT still suppresses Ca2+ influx in JCT8 cells. Increase in inositol phosphates by CD3 stimulation of JCT8 cells, including of inositol 1,4,5-triphosphate (I(1,4,5)P3), is only partially antagonized by CT. This suggests either that in JCT8 cells there is a different susceptibility of Ca2+ mobilization and influx to partial inhibition by CT of CD3-triggered phospholipase C (PLC)-induced phosphoinositide hydrolysis or that an additional and PLC-independent suppressive effect of the toxin on Ca2+ influx may exist. To investigate this particular point further, we use Thapsigargin, a Ca(2+)-endoplasmic reticulum ATPase inhibitor that can mobilize in human T lymphocytes I(1,4,5)P3-dependent intracellular Ca2+ pools by a PLC-independent pathway. We demonstrate that the Ca2+ influx triggered in the wild-type Jurkat cells or in JCT8 cells by Thapsigargin is antagonized by CT. The present data are therefore consistent with the idea that CT specifically impairs in the Jurkat T cell model the entry of Ca2+ from extracellular spaces by a mechanism independent not only from cAMP but also in part from inhibition by the toxin of phosphoinositide hydrolysis.
...
PMID:Cyclic AMP- and inositol phosphate-independent inhibition of Ca2+ influx by cholera toxin in CD3-stimulated Jurkat T cells. A study with a cholera toxin-resistant cell variant and the Ca2+ endoplasmic reticulum-ATPase inhibitor thapsigargin. 165 Mar 86

In rat luteal cells, an increase in intracellular [Ca]i impairs luteal function similar to that of prostaglandin F2a (PGF2a). However, calcium per se is not the mediator of the antigonadotropic action of PGF2a. Thapsigargin, a plant sesquiterpene lactone, increases intracellular calcium concentration concentration ([Ca]i) in several cell types by a mechanism that involves specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase. To further investigate the antigonadotropic role of [Ca]i and the mechanism of action of PGF2a in rat luteal cells, the action of thapsigargin on cellular functional responses was examined in the absence and presence of PGF2a. Thapsigargin dose dependently increased [Ca]i and inhibited cAMP accumulation and progesterone production in response to LH. The inhibitory effect of thapsigargin on cAMP accumulation was calcium dependent but in contrast, inhibition of LH-stimulated progesterone production was independent of calcium mobilization by thapsigargin. Steroidogenesis stimulated by (Bu)2cAMP was also inhibited by thapsigargin. Thus, thapsigargin mimicked some effects of PGF2a with inhibitory sites of action on both cAMP accumulation and progesterone production. Thapsigargin also blocked the mobilization of [Ca]i by PGF2a, but when coincubated with PGF2a an additive effect on inhibition of LH-stimulated progesterone production occurred. However, no additive effects of thapsigargin and PGF2a on gonadotropin-sensitive cAMP accumulation were evident. In conclusion, although thapsigargin and PGF2a may share some similar actions, their antigonadotropic effects are mediated differently.
...
PMID:The calcium-mobilizing agent, thapsigargin, inhibits progesterone production in rat luteal cells by a calcium-independent mechanism. 169 48

Thapsigargin, a non-phorbol-ester-type tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca(2+)-ATPase. We used this drug to analyze the involvement of Ca2+ and Ca(2+)-ATPases in the control of growth- and transformation-related genes. Here we show that treatment of mouse NIH 3T3 fibroblasts with thapsigargin induced rapid expression of the c-fos and c-jun protooncogenes. Inhibition or depletion of protein kinase C partially diminished the c-fos but not the c-jun response. Furthermore, thapsigargin could synergize with the tumor promoter phorbol 12-myristate 13-acetate to induce c-fos but not c-jun. However, thapsigargin had no effect on basal or phorbol ester-induced protein kinase C activity. Our results indicate that Ca2+ is a potent second messenger that controls expression of growth- and transformation-related genes. Since inhibition of the endoplasmic reticulum Ca(2+)-ATPase results in a strong induction of these genes, our data suggest that this Ca2+ pump may act as a negative regulator of cell growth.
...
PMID:Regulation of c-fos and c-jun protooncogene expression by the Ca(2+)-ATPase inhibitor thapsigargin. 171 85

Activation of a wide variety of membrane receptors leads to a sustained elevation of intracellular Ca2+ ([Ca2+]i) that is pivotal to subsequent cell responses. In general, in nonexcitable cells this elevation of [Ca2+]i results from two sources: an initial release of Ca2+ from intracellular stores followed by an influx of extracellular Ca2+. These two phases, release from intracellular stores and Ca2+ influx, are generally coupled: stimulation of influx is coordinated with depletion of Ca2+ from stores, although the mechanism of coupling is unclear. We have previously shown that histamine effects a typical [Ca2+]i response in interphase HeLa cells: a rapid rise in [Ca2+]i followed by a sustained elevation, the latter dependent entirely on extracellular Ca2+. In mitotic cells only the initial elevation, derived by Ca2+ release from intracellular stores, occurs. Thus, in mitotic cells the coupling of stores to influx may be specifically broken. In this report we first provide additional evidence that histamine-stimulated Ca2+ influx is strongly inhibited in mitotic cells. We show that efflux is also strongly stimulated by histamine in interphase cells but not in mitotics. It is possible, thus, that in mitotics intracellular stores are only very briefly depleted of Ca2+, being replenished by reuptake of Ca2+ that is retained within the cell. To ensure the depletion of Ca2+ stores in mitotic cells, we employed the sesquiterpenelactone, thapsigargin, that is known to affect the selective release of Ca2+ from intracellular stores by inhibition of a specific Ca(2+)-ATPase; reuptake is inhibited. In most cells, and in accord with Putney's capacitative model (1990), thapsigargin, presumably by depleting intracellular Ca2+ stores, stimulates Ca2+ influx. This is the case for interphase HeLa cells. Thapsigargin induces an increase in [Ca2+]i that is dependent on extracellular Ca2+ and is associated with a strong stimulation of 45Ca2+ influx. In mitotic cells thapsigargin also induces a [Ca2+]i elevation that is initially comparable in magnitude and largely independent of extracellular Ca2+. However, unlike interphase cells, in mitotic cells the elevation of [Ca2+]i is not sustained and 45Ca2+ influx is not stimulated by thapsigargin. Thus, the coupling between depletion of intracellular stores and Ca2+ influx is specifically broken in mitotic cells. Uncoupling could account for the failure of histamine to stimulate Ca2+ influx during mitosis and would effectively block all stimuli whose effects are mediated by Ca2+ influx and sustained elevations of [Ca2+]i.
...
PMID:Regulation of Ca2+ influx during mitosis: Ca2+ influx and depletion of intracellular Ca2+ stores are coupled in interphase but not mitosis. 180 98


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---