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)

In the present paper, P1 and P2 purinergic receptors and their control of signal transduction pathways were investigated in NCB-20 cells. ATP elicited an increase in [Ca2+]i. The purinergic receptor subtype involved was identified by comparing the actions of a range of nucleotides. UTP was the most potent agonist in elevating [Ca2+]i, with an EC50 value of 6.2 +/- 0.5 microM. UTP, ATP (EC50, 17.3 +/- 1.5 microM), adenosine-5'-O-(3-thio)triphosphate (23 +/- 3 microM), and ITP (55 +/- 4 microM) exerted similar maximal effects. Other nucleotides tested, including beta, gamma-methylene-ATP and 2-methylthio-ATP, which are considered prototypic agonists for P2x and P2y receptors, respectively, were ineffective; in general, modifications in the ribose-triphosphate chain and substitution on the 2-position of the purines reduced the efficacy of nucleotides. This pharmacological characterization indicated that a putative P2u receptor mediates the [Ca2+]i elevation elicited by nucleotides in NCB-20 cells. The increase in [Ca2+]i originates from intracellular Ca2+ stores; blockade of Ca2+ entry does not affect the rise in [Ca2+]i. In contrast, pretreatment with the Ca(2+)-ATPase inhibitor thapsigargin or with bradykinin, a hormone that releases Ca2+ from inositol trisphosphate-sensitive stores, does preclude the increase in [Ca2+]i induced by ATP. ATP and UTP also transiently inhibit cAMP accumulation in the intact cell, presumably via a Ca(2+)-mediated mechanism. The finding of a P2u receptor in NCB-20 cells adds to a growing perception that P2 receptors are widely distributed. Besides the P2u receptor, NCB-20 cells express adenosine A2 receptors, coupled to stimulation of cAMP accumulation. The presence of both P1 and P2 purinergic receptors permits a sequential modulation of distinct second messenger levels associated with a common stimulus, ATP.
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PMID:Purinergic receptor regulation of signal transduction in NCB-20 cells. 131 45

In rabbits the contractile response of the urinary bladder is only partially due to cholinergic innervation since atropine does not completely block neuronally mediated contractions. In the human bladder this atropine resistance is controversial with some reporting atropine resistance in vitro while others have stated that the atropine resistance is also tetrodotoxin resistant. Results of the present investigation demonstrate that an atropine resistant, tetrodotoxin sensitive contraction can be evoked in some, but not all human bladder strips. Evidence accumulated over the past few decades indicates that this atropine resistant contraction may be mediated by ATP or a related purine compound. Studies presented herein are designed to develop a radioligand assay for this purinergic receptor. Initial studies indicated that the hydrolysis resistant ATP analog beta, gamma methylene ATP offers several advantages over ATP as a potential radioligand. It is only slowly hydrolyzed by endogenous ATPase and does not inhibit the hydrolysis of ATP indicating that it probably does not bind to the active sites of endogenous ATP hydrolyzing enzymes. In addition beta, gamma methylene ATP is 10-100 fold more potent than ATP itself in stimulating contractions of the urinary bladder in-vitro. The radioligand binding assay herein described can be used to quantitate the density of purinergic receptors, an essential step for determining the role of this system in urinary bladder function and dysfunction. Application of this assay could form the foundation for development of a new class of therapeutic agents for the treatment of urinary bladder dysfunction based on modulation of the purinergic nervous system.
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PMID:Bladder purinergic receptors. 214 55

Stable Chinese hamster ovary (CHO) cell lines overexpressing the human plasma membrane Ca(2+)-ATPase (PMCA) were generated, and three independent cell clones were characterized in details. They overexpressed high amounts of active PMCA pump (15-20 times over the amount of endogenous PMCA) as indicated by experiments in which the formation of the phosphoenzyme intermediate and the uptake of Ca2+ by microsomes were measured. Immunocytochemistry experiments coupled to the biotinylation of the pump in the intact cells indicated the correct deliver of the expressed pump to the plasma membrane. The expressed pump was purified by affinity chromatography on calmodulin sepharose. The PMCA of transfected CHO cells promoted an increase of Ca2+ into the medium, after induction of Ca2+ release from the internal stores by activation of a purinergic receptor. An evident decrease of the activity of the endogenous sarcoplasmic reticulum Ca(2+)-ATPase pump was observed, probably related to the down-regulation of its expression. The cells overexpressing the PMCA pump had delayed recovery after trypsinization and plating. Their doubling time was, however, the same as CHO cells.
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PMID:Isolation and characterization of a stable Chinese hamster ovary cell line overexpressing the plasma membrane Ca(2+)-ATPase. 778 27

A microfluorimetric method using Fura-2 as calcium indicator was used to study the mechanism of desensitization of the calcium response evoked by activation of a brain endothelial cell P2U receptor. The study was mainly carried out on an immortalized rat brain endothelial cell line (RBE4), with some additional experiments on primary cultured rat brain microvascular endothelial cells. As previously described (Nobles et al. 1995), ATP (100 microM, 20 s) caused a transient increase in intracellular calcium levels ([Ca2+]i). This effect was dependent on the rate of filling of intracellular calcium stores, since a large inhibition of the ATP-mediated response was seen in the presence of cyclopiazonic acid, an inhibitor of the store Ca(2+)-ATPase. Application of repeated pulses of extracellular ATP led to a desensitization of the response, as measured by a decline in the release of intracellular calcium (Nobles et al. 1995). This desensitization was partially reversed after 300 s of incubation in agonist-free medium. Extracellular phosphorylation of the purinergic receptor appeared not to be involved in the desensitization process, since a similar rate of desensitization was obtained with the non-hydrolysable ATP analogue ATP gammaS. Oxidation of the purinergic receptor cannot account for the desensitization, since the decline of the ATP-mediated response was unchanged in the presence of 3 mM dithiothreitol. In the presence of ATP together with UTP, two equally potent activators of the P2U receptor, the desensitization was less than in the presence of only one of the agonists. The desensitization was greater when ATP was applied for longer (150 s) periods. Although these results do not exclude the participation of post-receptor events in the desensitization process, they suggest that desensitization is governed at least in part by agonist-receptor interaction.
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PMID:Decline of the calcium response on successive stimulation of a rat brain endothelial cell P2U purinoceptor. 949 4

The influence of cooling on the intracellular concentration of Ca2+ ([Ca2+]i) was tested in cell lines expressing chemical receptors. First, when ATP was externally added to rat basophilic leukemia (RBL-2H3) cells, cooling from 37 degrees C to 27 degrees C induced a transient rapid increase in [Ca2+]i. In the absence of extracellular Ca2+, the [Ca2+]i response was induced whereas an inhibitor of microsomal Ca2+ ATPase, thapsigargin, largely abolished the [Ca2+]i response, suggesting that the internal Ca2+ store liberate the Ca2+. A purinergic receptor antagonist, suramin, completely inhibited the [Ca2+]i response to the cooling. Secondly, when serotonin (5-HT) was added to rat glioma C6BU-1 cells, the cooling induced a transient increase in [Ca2+]. This [Ca2+]i response was induced in the absence of external Ca2+, suggesting that the internal Ca2+ stores liberate the Ca2+. These results raise the possibility that some G protein-coupled receptors are sensitive to cooling in the presence of agonist for the receptor.
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PMID:Cooling sensitive [Ca2+]i response associated with signaling of G protein-coupled receptors. 970 96

alpha-Sarcoglycan is a component of the sarcoglycan complex of dystrophin-associated proteins. Mutations of any of the sarcoglycan genes cause specific forms of muscular dystrophies, collectively termed sarcoglycanopathies. Importantly, a deficiency of any specific sarcoglycan affects the expression of the others. Thus, it appears that the lack of sarcoglycans deprives the muscle cell of an essential, yet unknown function. In the present study, we provide evidence for an ecto-ATPase activity of alpha-sarcoglycan. alpha-Sarcoglycan binds ATP in a Mg2+-dependent and Ca2+-independent manner. The binding is inhibited by 3'-O-(4-benzoyl)benzoyl ATP and ADP. Sequence analysis reveals the existence of a consensus site for nucleotide binding in the extracellular domain of the protein. An antibody against this sequence inhibits the binding of ATP. A dystrophin.dystrophin-associated protein preparation demonstrates a Mg-ATPase activity that is inhibited by the antibody but not by inhibitors of endo-ATPases. In addition, we demonstrate the presence in the sarcolemmal membrane of a P2X-type purinergic receptor. These data suggest that alpha-sarcoglycan may modulate the activity of P2X receptors by buffering the extracellular ATP concentration. The absence of alpha-sarcoglycan in sarcoglycanopathies leaves elevated the concentration of extracellular ATP and the persistent activation of P2X receptors, leading to intracellular Ca2+ overload and muscle fiber death.
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PMID:Ecto-ATPase activity of alpha-sarcoglycan (adhalin). 1007 85

Human leukocytes can express the P2X(7) purinergic receptor, an ionic channel gated by extracellular ATP, for which the physiological role is only partially understood. Transfection of P2X(7) cDNA into lymphoid cells that lack this receptor sustains their proliferation in serum-free medium. Increased proliferation of serum-starved P2X(7) transfectants is abolished by the P2X(7) receptor blocker oxidized ATP or by the ATP hydrolase apyrase. Both wild type and P2X(7)-transfected lymphoid cells release large amounts of ATP into the culture medium. These data suggest the operation of an ATP-based autocrine/paracrine loop that supports lymphoid cell growth in the absence of serum-derived growth factors.
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PMID:Increased proliferation rate of lymphoid cells transfected with the P2X(7) ATP receptor. 1055 92

Intercellular Ca(2+)-signaling, after mechanical stimulation of calf pulmonary artery endothelial cells (CPAE), was investigated with fluorescence video imaging. Mechanical stimulation evoked an intracellular Ca(2+)-response in the mechanically stimulated (MS) cell, proceeding to the neighboring (NB) cells as a Ca(2+)-wave. The intercellular propagation of the Ca(2+)-wave was unaffected by the gap junction blockers halothane or heptanol. Therefore the intercellular communication (IC) pathway of the Ca(2+)-wave in CPAE cells does not depend on gap junctional communication but is most likely mediated by release of an extracellular mediator. Continuous unilateral flow experiments confirmed the presence of a diffusible mediator: the Ca(2+)-rise in upstream NB cells is significantly lower than in control experiments. After desensitization of purinergic receptors by pretreatment of CPAE cells with ATP (100mM), UTP (100 microM), 2MeSATP (100microM) or ADPbS (100 microM), the propagation of the intercellular Ca(2+)-wave upon mechanical stimulation was significantly inhibited. Also suramin (200 and 400 microM), a non-specific purinergic receptor blocker, reduced the IC. Application of the nucleotidase apyrase VI (10U/ml), which has a high ATPase/ADPase ratio, enhanced Ca(2+)-signaling and IC. In contrast, apyrase VII (10U/ml), which has a high ADPase/ATPase ratio, significantly depressed the propagation of the intercellular Ca(2+)-wave upon mechanical stimulation. Our experiments therefore demonstrate that the IC, evoked by a mechanical stimulus of CPAE cells, is mediated via release of nucleotides in the extracellular space. The data indicate that the diffusible messenger, responsible for the propagation of a Ca(2+)-wave, is mainly ADP or a combination of ADP/ATP.
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PMID:Intercellular communication upon mechanical stimulation of CPAE- endothelial cells is mediated by nucleotides. 1116 50

The mechanisms involved in autocrine ATP release from cultured astrocytes isolated from the rat cortex were investigated using an online bioluminescence technique. Astrocytes released ATP in response to application of 10 microM uridine triphosphate, which was blocked by the non-specific purinergic receptor antagonist suramin. Intracellular pathways of the uridine triphosphate-stimulated ATP release were seen to involve inositol triphosphate and calcium with the assistance of the Golgi-complex and cytoskeleton as the release was inhibited by phospholipase C antagonist lithium, endoplasmic reticulum calcium-dependent ATPase inhibitor thapsigargin, F-actin interruptor cytochalasin D and Golgi-complex interruptor brefeldin A. The uridine triphosphate-stimulated ATP release was also potently blocked by exocytosis inhibitor botulinum toxin A and anion transporter blockers furosemide and glibenclamide. These results suggest that calcium-dependent exocytosis and transportation via anion transporters are the predominant secretion mechanisms for uridine triphosphate-stimulated ATP release from cortical astrocytes.
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PMID:Mechanisms of secretion of ATP from cortical astrocytes triggered by uridine triphosphate. 1462 43

The ciliary beat frequency (CBF) of rat tracheal ciliary cells in a slice preparation was measured using video-enhanced contrast (VEC) microscopy. Acetylcholine (ACh) increased CBF mediated via intracellular Ca2+ concentration ([Ca2+]i) in a dose-dependent manner. An adequate hypo-osmotic stress (-40 mosM) potentiated ACh-stimulated CBF increase in tracheal ciliary cells and shifted the ACh dose-response curve to the left (lower concentration side). This potentiation was independent of hypo-osmotic stresses applied ranging from -20 mosM to -90 mosM. A hypo-osmotic stress induces ATP release in many cell types. The present study demonstrated that suramin (an inhibitor of purinergic receptors) and apyrase (an ATPase/ADPase) eliminate the hypo-osmotic potentiation of ACh-stimulated CBF increase and that ATP increased [Ca2+]i and CBF, as well as potentiating ACh-stimulated rises in [Ca2+]i and CBF increase. Moreover, the apical surface of tracheal ciliary cells were stained immunopositive for the P2X4 purinergic receptor. A hypo-osmotic stress (-40 mosM) transiently increased [Ca2+]i and potentiated the ACh-stimulated [Ca2+]i increase. The hypo-osmotic potentiation of ACh-stimulated CBF increase was not detected under Ca2+-free conditions. These observations suggest that a hypo-osmotic stress stimulates ATP release from the trachea. The released ATP may induce further increases in [Ca2+]i and CBF in ACh-stimulated tracheal ciliary cells, which may be mediated by purinergic receptors, such as P2X4.
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PMID:Hypo-osmotic potentiation of acetylcholine-stimulated ciliary beat frequency through ATP release in rat tracheal ciliary cells. 1536 81


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