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:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

Stimulation of Ca2+ (and Mn2+) entry in salivary epithelial cells by carbachol, or thapsigargin, is mediated by an, as yet, unknown mechanism that is dependent on the depletion of Ca2+ from intracellular Ca2+ stores. This study assesses the possible role of protein phosphorylation in the regulation of Ca2+ entry in rat parotid gland acinar cells. Treatment of cells with the protein phosphatase inhibitors okadaic acid, calyculin A, and pervanadate induced a dose-dependent inhibition of carbachol and thapsigargin stimulation of Ca2+ and Mn2+ entry. All three inhibitors decreased carbachol stimulation of internal Ca2+ release, which likely accounts for the inhibition of carbachol-stimulated Ca2+ entry. Thapsigargin-induced internal Ca2+ release was not affected by the treatments. Additionally, all three phosphatase inhibitors decreased Mn2+ entry into cells with depleted internal Ca2+ store(s) (achieved by incubation with either carbachol or thapsigargin in Ca2+-free medium). Treatment of cells with phorbol 12-myristate 13-acetate, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, or staurosporine did not affect divalent cation entry into unstimulated cells or thapsigargin treated cells. Importantly, when cells with depleted internal Ca2+ store(s) were pretreated with staurosporine, or K-252a, the inhibition of Ca2+ entry by calyculin A and okadaic acid, but not by pervanadate, was attenuated. Although the effect of pervanadate remains to be clarified, these results demonstrate a role for protein phosphorylation in the regulation of divalent cation influx in rat parotid acinar cells.
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PMID:Role for protein phosphatase in the regulation of Ca2+ influx in parotid gland acinar cells. 876 57

Cytosolic Ca2+ concentration ([Ca2+]i) plays an important role in control of pulmonary vascular endothelial cell (ECs) barrier function. In this study, we investigated whether thapsigargin- and ionomycin-induced changes in cytosolic Ca2+ induce permeability in rat pulmonary microvascular (RPMV) versus macrovascular (RPA) ECs. In Transwell cultures, RPMVECs formed a tighter, more restrictive barrier than RPAECs to 12,000-, 72,000-, and 150,000-molecular-weight FITC-labeled dextrans. Thapsigargin (1 microM) produced higher [Ca2+]i levels in RPAECs than in RPMVECs and increased permeability in RPAEC but not in RPMVEC monolayers. Due to the attenuated [Ca2+]i response in RPMVECs, we investigated whether reduced activation of store-operated Ca2+ entry was responsible for the insensitivity to thapsigargin. Addition of the drug in media containing 100 nM extracellular Ca2+ followed by readdition media with 2 mM extracellular Ca2+ increased RPMVEC [Ca2+]i to a level higher than that in RPAECs. Under these conditions, RPMVEC permeability was not increased, suggesting that [Ca2+]i in RPMVECs does not initiate barrier disruption. Also, ionomycin (1.4 microM) did not alter RPMVEC permeability, but the protein phosphatase inhibitor calyculin A (100 nM) induced permeability in RPMVECs. These data indicate that, whereas increased [Ca2+]i promotes permeability in RPAECs, it is not sufficient in RPMVECs, which show an apparent uncoupling of [Ca2+]i signaling pathways or dominant Ca(2+)-independent mechanisms from controlling cellular gap formation and permeability.
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PMID:Pulmonary microvascular and macrovascular endothelial cells: differential regulation of Ca2+ and permeability. 961 97

We studied spiking neurons isolated from turtle retina by the whole cell version of the patch clamp. The studied cells had perikaryal diameters > 15 microns and fired multiple spikes in response to depolarizing current steps, indicating they were ganglion cells. In symmetrical [Cl-], currents elicited by puffs of 100 microM gamma-aminobutyric acid (GABA) were inward at a holding potential of -80 mV. All of the GABA-evoked current was blocked by SR95331 (20 microM), indicating that it was mediated by a GABAA receptor. The GABA-evoked currents were unaltered by eliciting a transmembrane calcium current either just before or during the response to GABA. On the other hand caffeine (10 mM), which induces Ca2+ release from intracellular stores, inhibited the GABA-evoked current on average by 30%. The caffeine effect was blocked by introducing the calcium buffer bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) into the cell but was unaffected by replacing [Ca2+]o with equimolar cobalt. Thapsigargin (10 microM), an inhibitor of intracellular calcium pumps, and ryanodine (20 microM), which depletes intracellular calcium stores, both markedly reduced a caffeine-induced inhibition of the GABA-evoked current. Another activator of intracellular calcium release, inositol trisphosphate (IP3; 50 microM), also progressively reduced the GABA-induced current when introduced into the cell. Dibutyryl adenosine 3'5'-cyclic monophosphate (cAMP; 0.5 mM), a membrane-permeable analogue of cAMP, did not reduce GABA-evoked currents, suggesting that cAMP-dependent kinases are not involved in suppressing GABAA currents, whereas calmidazolium (30 microM) and cyclosporin A (20 microM), which inhibit Ca/calmodulin-dependent phosphatases, did reduce the caffeine-induced inhibition of the GABA-evoked current. Alkaline phosphatase (150 micrograms/ml) and calcineurin (300 micrograms/ml) had a similar action to caffeine or IP3. Antibodies directed against the ryanodine receptor or the IP3 receptor reacted with the great majority of neurons in the ganglion cell layer. We found that these two antibodies colocalized in large ganglion cells. In summary, intracellular calcium plays a role in reducing the currents elicited by GABA, acting through GABAA receptors. The modulatory action of calcium on GABA responses appears to work through one or more Ca-dependent phosphatases.
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PMID:Calcium released from intracellular stores inhibits GABAA-mediated currents in ganglion cells of the turtle retina. 974 25

IL-8, a potent neutrophil chemoattractant that is elevated about 200-fold in exudative neutrophils isolated from localized inflammatory sites in vivo, is thought to play a major role in recruitment of neutrophils to inflammatory sites. Incubation of peripheral blood neutrophils with thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-sequestering-ATPase, causes a dose-dependent induction of IL-8 synthesis that continues for up to 8 h. Cycloheximide inhibits the thapsigargin-induced IL-8 production, suggesting the induction of protein synthesis de novo. In addition, Northern blot analysis of mRNA isolated from neutrophils indicates that thapsigargin treatment increases IL-8 mRNA in a time- and dose-dependent manner. Thapsigargin also induces a biphasic rise in the intracellular Ca2+ concentration, [Ca2+]i, which is composed of an initial (within 15 s) EGTA-insensitive elevation in [Ca2+]i, followed by a delayed (2-min) EGTA-sensitive component. Addition of EGTA before thapsigargin inhibited the induction of IL-8 production. Experiments in which EGTA was added at various times after thapsigargin treatment indicated that a sustained Ca2+ influx was required for maximum IL-8 production. Ascomycin and cyclosporin A, inhibitors of the Ca2+-dependent phosphatase, calcineurin, also inhibited thapsigargin-induced IL-8 production. Thus, in neutrophils, a prolonged increase in [Ca2+]i stimulates IL-8 transcription and synthesis, possibly through a calcineurin-dependent pathway.
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PMID:Ca2+-dependent production and release of IL-8 in human neutrophils. 978 Feb 10

We have shown previously that calcium ionophore (CI) treatment of various myeloid origin cells results in rapid acquisition of properties associated with mature, activated dendritic cells. These properties include increased CD83 and costimulatory molecule expression, tendencies to form dendritic processes, loss of CD14 expression by monocytes, and typically an enhanced capacity to sensitize T lymphocytes to Ag. We here analyze the intracellular signaling pathways by which CI induces acquisition of such properties. Thapsigargin, which raises intracellular Ca2+ levels by antagonizing its sequestration, induced immunophenotypic and morphologic changes that paralleled CI treatment. CI-induced activation was broadly attenuated by the Ca2+ chelating compound EGTA and by calmodulin antagonists trifluoperazine dimaleate and W-7. However, antagonists of signaling pathways downstream to calmodulin displayed more selective inhibitory effects. Calcineurin antagonists cyclosporin A and the FK-506 analogue, ascomycin, diminished costimulatory molecule and CD83 expression, as well as formation of dendritic processes in CI-treated myeloid cells, and strongly attenuated the T cell allosensitizing capacity of CI-treated HL-60 cells. These calcineurin antagonists displayed minimal effect on CI-induced CD14 down-regulation in monocytes. In contrast, the calmodulin-dependent protein kinase antagonists, K252a and KT5926, while displaying only modest effects on CI-induced costimulatory molecule and CD83 expression, strongly blocked CD14 down-regulation. These results are consistent with a Ca2+-dependent mechanism for CI-induced differentiation of myeloid cells, and indicate that multiple discrete signaling pathways downstream to calcium mobilization and calmodulin activation may be essential in regulating this process.
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PMID:Calcium mobilization in human myeloid cells results in acquisition of individual dendritic cell-like characteristics through discrete signaling pathways. 1038 3

Nicotinic acetylcholine receptors containing alpha7 subunits have a high relative permeability to calcium and influence numerous calcium-dependent cellular events. On chick ciliary ganglion neurons the receptors are concentrated on somatic spines containing actin filaments. Using conventional whole-cell patch-clamp recording from dissociated ciliary ganglion neurons, we show that responses from alpha7-containing receptors undergo substantial rundown when the receptors are repeatedly challenged with nicotine. Stabilization of actin filaments with phalloidin partially prevents the rundown, whereas collapse of actin filaments with latrunculin A exacerbates it. The rundown depends on calcium influx through the receptors because it requires receptor activation and can be prevented by replacing extracellular calcium with barium or by intracellular dialysis with BAPTA. Thapsigargin and ryanodine each inhibit the rundown, demonstrating further a requirement for calcium release from internal stores. Blockade of calmodulin by calmidazolium or blockade of CaM kinase II with either KN93 or autocamtide-2-related inhibitory peptide each prevents the rundown; blockade of the phosphatase calcineurin with either cyclosporin A or deltamethrin increases the rundown. The results indicate a balance of calcium-dependent kinase and phosphatase activities in regulating the function of alpha7-containing receptors. Manifestation of the rundown depends in part on the loss of intracellular components via dialysis because little rundown is seen if perforated patch-clamp recording is used to monitor receptor responses even in latrunculin A-treated cells. A membrane-permeable calcineurin inhibitor, however, still decreases the nicotinic response in a calcium-dependent manner, confirming that calcium-dependent phosphoregulation of alpha7-containing receptors occurs in the intact cell.
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PMID:Actin filaments and the opposing actions of CaM kinase II and calcineurin in regulating alpha7-containing nicotinic receptors on chick ciliary ganglion neurons. 1057 25

Thapsigargin (TG), which inhibits endoplasmic reticulum-dependent Ca(2 +)-ATPase and thereby increases cytosolic Ca(2 +), has been reported to cause apoptosis in T lymphocytes another cell types. In this study, we investigated the molecular mechanisms that are involved in the apoptosis induced by TG in T cell hybridomas. Exposure to TG results in rapid induction of the orphan steroid receptor, Nur77, accompanied by apoptosis of T cell hybridomas. The expression of Nur77 in response to TG treatment is sensitive to cyclosporin A, implicating that activation of calcineurin is necessary for Nur77 expression. The TG-induced Nur77 expression is also inhibited by overexpression of Cabin1, an endogenous inhibitor of calcineurin and a corepressor of the transcription factor MEF2, suggesting that MEF2 activation is required for Nur77 expression. These results suggest that induction of Nur77 expression and apoptosis by TG are mediated by the same signaling pathways that are involved in T cell receptor-mediated thymocyte apoptosis, including the calcineurin pathway and Cabin1-MEF2 pathway.
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PMID:Thapsigargin-induced apoptosis involves Cabin1-MEF2-mediated induction of Nur77. 1138 20

We have investigated cellular Ca2+ regulation during A2058 human melanoma cell chemotaxis to type IV collagen (CIV). We have identified alpha2beta1-integrin as the primary mediator of A2058 cell response to CIV in vitro. Integrin ligation initiated a characteristic intracellular Ca2+ concentration ([Ca2+]i) response consisting of an internal release and a receptor-mediated Ca2+ entry. Thapsigargin (TG) pretreatment drained overlapping and CIV-inducible internal Ca2+ stores while initiating a store-operated Ca2+ release (SOCR). CIV-mediated Ca2+ entry was additive to TG-SOCR, suggesting an independent signaling mechanism. Similarly, ionophore application in a basal medium containing Ca2+ initiated a sustained influx. Elevated [Ca2+]i from TG-SOCR or ionophore significantly attenuated cell migration to CIV by recruiting the Ca2+/calcineurin-mediated signaling pathway. Furthermore, low [Ca2+]i induced by EGTA application in the presence of ionophore fully restored cell motility to CIV. Together, these results suggest that [Ca2+]i signaling accompanying A2058 cell response to alpha2beta1-integrin ligation is neither necessary nor sufficient and that elevated [Ca2+]i downregulates cell motility via a calcineurin-mediated mechanism in A2058 cell chemotaxis to CIV.
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PMID:[Ca2+]i as a potential downregulator of alpha2beta1-integrin-mediated A2058 tumor cell migration to type IV collagen. 1140 32

Stress of the endoplasmic reticulum (ER), which is associated with many neurodegenerative conditions, can lead to the elimination of affected cells by apoptosis through only partially understood mechanisms. Thapsigargin, which causes ER stress by inhibiting the ER Ca(2+)-ATPase, was found to not only activate the apoptosis effector caspase-3 but also to cause a large and prolonged increase in the activity of glycogen synthase kinase-3beta (GSK3beta). Activation of GSK3beta was obligatory for thapsigargin-induced activation of caspase-3, because inhibition of GSK3beta by expression of dominant-negative GSK3beta or by the GSK3beta inhibitor lithium blocked caspase-3 activation. Thapsigargin treatment activated GSK3beta by inducing dephosphorylation of phospho-Ser-9 of GSK3beta, a phosphorylation that normally maintains GSK3beta inactivated. Caspase-3 activation induced by thapsigargin was blocked by increasing the phosphorylation of Ser-9-GSK3beta with insulin-like growth factor-1 or with the phosphatase inhibitors okadaic acid and calyculin A, but the calcineurin inhibitors FK506 and cyclosporin A were ineffective. Insulin-like growth factor-1, okadaic acid, calyculin A, and lithium also protected cells from two other inducers of ER stress, tunicamycin and brefeldin A. Thus, ER stress activates GSK3beta through dephosphorylation of phospho-Ser-9, a prerequisite for caspase-3 activation, and this process is amenable to pharmacological intervention.
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PMID:Central role of glycogen synthase kinase-3beta in endoplasmic reticulum stress-induced caspase-3 activation. 1222 24


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