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1
Pastorino, J. G., J. W. Snyder, J. B. Hoek, and J. L. Farber. "Ca2+ depletion prevents anoxic death of hepatocytes by inhibiting mitochondrial permeability transition." American Journal of Physiology-Cell Physiology 268, no. 3 (March 1, 1995): C676—C685. http://dx.doi.org/10.1152/ajpcell.1995.268.3.c676.
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Removal of Ca2+ from the culture medium or treatment with the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) prevented the killing of rat hepatocytes by anoxia and rotenone, but not by cyanide. Neither manipulation prevented the loss of the mitochondrial membrane potential or the depletion of ATP. A mitochondrial permeability transition (MPT) was demonstrated in digitonin-permeabilized hepatocytes as an increased [3H]sucrose-accessible space sensitive to cyclosporin A (CyA). Ca2+ depletion by either means prevented the MPT measured in intact cells made anoxic or treated with rotenone. In isolated mitochondria deenergized by rotenone, BAPTA-AM prevented the MPT induced by palmitoyl CoA. By contrast, in isolated mitochondria deenergized by cyanide, BAPTA-AM alone did not prevent the MPT. Rather, BAPTA-AM plus CyA were required. Similarly, the killing of cultured hepatocytes by cyanide was prevented by BAPTA-AM plus CyA, but not by either agent alone. The MPT in intact cells treated with cyanide was also prevented by BAPTA-AM plus CyA. These data define a specific requirement for Ca2+ in the killing of hepatocytes that follows the inhibition of electron transport. A model is presented in which the MPT depends on factors that modulate the sensitivity of the permeability transition to the matrix concentration of Ca2+.
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Kuijpers, T. W., M. Hoogerwerf, and D. Roos. "Neutrophil migration across monolayers of resting or cytokine-activated endothelial cells. Role of intracellular calcium changes and fusion of specific granules with the plasma membrane." Journal of Immunology 148, no. 1 (January 1, 1992): 72–77. http://dx.doi.org/10.4049/jimmunol.148.1.72.
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Abstract Chemoattractants, used at concentrations to invoke optimal neutrophil chemotaxis, induce rapid changes in neutrophils such as a transient increase in intracellular Ca2+ ([Ca2+]i). We have previously observed that neutrophils adhering to cytokine-activated endothelial cells (EC) also respond with a rapid rise in [Ca2+]i caused by an endothelial membrane-bound form of platelet-activating factor. After preloading with the intracellular Ca(2+)-chelator bis-(O-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid (BAPTA/AM), neutrophils were no longer able to respond with a rapid rise in [Ca2+]i toward the chemoattractant FMLP or to rIL-1 beta-pretreated EC. These neutrophils were still able to adhere and migrate under the conditions tested. The only difference was that the BAPTA/AM-treated neutrophils migrated a little slower than untreated control neutrophils. This discrepancy was not observed at later time points. The BAPTA/AM-preloaded neutrophils did not differ from unloaded neutrophils in actin polymerization responses. Whereas untreated neutrophils demonstrated an up-regulation of the specific granule markers CD11b, CD45, and CD67 during migration (without any release from the azurophil granules), the BAPTA/AM pretreatment completely prevented this process. The BAPTA/AM-preloaded neutrophils did not release vitamin B12-binding protein from the specific granules upon treatment with FMLP. The down-modulation of the selectin member LAM-1, as seen upon neutrophil activation, was not affected by BAPTA/AM pretreatment of the neutrophils. Thus, neither the rapid rise in [Ca2+]i nor specific granule fusion with the plasma membrane constitute a prerequisite for neutrophil migration across resting or cytokine-activated EC.
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Billman, G. E. "Intracellular calcium chelator, BAPTA-AM, prevents cocaine-induced ventricular fibrillation." American Journal of Physiology-Heart and Circulatory Physiology 265, no. 5 (November 1, 1993): H1529—H1535. http://dx.doi.org/10.1152/ajpheart.1993.265.5.h1529.
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Cocaine is a potent cardiac stimulant that can provoke lethal cardiac events, including ventricular fibrillation (VF). The cocaine-induced accumulation of intracellular calcium could contribute significantly to the development of these lethal arrhythmias. To test this hypothesis, VF was induced in 12 mongrel dogs by the combination of cocaine (1.0 mg/kg) and a 2-min coronary occlusion during exercise. This test without cocaine failed to induce arrhythmias. Pretreatment with the intracellular calcium-specific chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM; 1.0 mg/kg iv) prevented VF in 8 of 12 animals (P < 0.001) and delayed the onset of lethal arrhythmias in 3 of the remaining animals. Cocaine induced significant increases in left ventricular (LV) systolic pressure (control 154.7 +/- 8.7, cocaine 167.4 +/- 8.4 mmHg), heart rate (control 195.9 +/- 6.1, cocaine 222.3 +/- 10.6 beats/min), and LV maximum rate of pressure development (dP/dtmax; control 5,251 +/- 317.6, cocaine 6,016 +/- 435.1 mmHg/s). BAPTA-AM attenuated the increase in LV dP/dtmax (BAPTA-AM 4,591 +/- 479.3 mmHg/s) and LV systolic pressure (BAPTA-AM 154.5 +/- 6.8 mmHg). Because vascular muscle relaxation could contribute to the cardioprotection, the cocaine and exercise plus ischemia test was repeated after nitroprusside. The nitroprusside prevented cocaine-induced increases in LV systolic pressure but failed to prevent VF. These data suggest that BAPTA-AM may prevent cocaine-induced VF independently of its vascular actions.
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Tymianski, Michael, Igor Spigelman, Liang Zhang, Peter L. Carlen, Charles H. Tator, Milton P. Charlton, and M. Christopher Wallace. "Mechanism of Action and Persistence of Neuroprotection by Cell-Permeant Ca2+ Chelators." Journal of Cerebral Blood Flow & Metabolism 14, no. 6 (November 1994): 911–23. http://dx.doi.org/10.1038/jcbfm.1994.122.
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Cell-permeant Ca2+ chelators such as 1,2-bis-(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM) have been reported to protect neurons in experimental focal cerebral ischemia. However, their in vivo actions are uncertain, and their protective efficacy is proven only in brief cerebral ischemia paradigms. Here we examine their mechanism of action in vitro and duration of efficacy in vivo. Electrophysiological studies were made in CA1 neurons in rat hippocampal slices. When superfused with BAPTA-AM (30–50 μ M), CA1 somatic field potential recordings showed attenuation of the population spike amplitude, and intracellular recordings showed reduced excitatory postsynaptic potentials, indicating inhibition of excitatory synaptic transmission. Also, Ca2+ -dependent accommodation and post-spike-train hyperpolarizations were reduced, indicating Ca2+ chelation hear the internal cell membrane surface. To determine whether Ca2+ chelators reduce the size of cerebral infarction rather than simply delaying its evolution, we studied the effects of BAPTA-AM treatment on infarction size 24 h after permanent middle cerebral artery occlusion. Fischer rats ( n = 8 per group) were pretreated with saline, BAPTA-AM (20 mg/kg), or MK-801 (0.5 mg/kg). Infarction volumes in animals treated with BAPTA-AM were reduced by 50.5% compared with controls ( p = 0.018), whereas animals treated with MK-801 experienced a statistically insignificant infarct volume reduction (26%; p = 0.27). These data show a persistence of neuroprotection by the Ca2+ chelator at 24 h and indicate that it may act by attenuating synaptic transmission and subplasma membrane Ca2+ excess.
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Liu, Qing, Ulrike Möller, Daniela Flügel, and Thomas Kietzmann. "Induction of plasminogen activator inhibitor I gene expression by intracellular calcium via hypoxia-inducible factor-1." Blood 104, no. 13 (December 15, 2004): 3993–4001. http://dx.doi.org/10.1182/blood-2004-03-1017.
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Abstract The plasminogen activator inhibitor-1 (PAI-1) expression can be enhanced by hypoxia and other stimuli leading to the mobilization of intracellular calcium. Thus, it was the aim of the present study to investigate the role of calcium in the hypoxia-dependent PAI-1 expression. It was shown that the Ca2+-ionophore A23187 and the cell permeable Ca2+-chelator BAPTA-am (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester) induced PAI-1 mRNA and protein expression under normoxia and hypoxia in HepG2 cells. Transfection experiments with wild-type and hypoxia response element (HRE)-mutated PAI promoter constructs revealed that the HRE binding hypoxiainducible factor-1 (HIF-1) mediated the response to A23187 and BAPTA-am. Although A23187 induced a striking and stable induction of HIF-1α, BAPTA-am only mediated a fast and transient increase. By using actinomycin D and cycloheximide we showed that A23187 induced HIF-1α mRNA expression, whereas BAPTA-am acted after transcription. Although A23187 activated extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK), as well as protein kinase B, it appeared that the enhancement of HIF-1α by A23187 was only mediated via the ERK pathway. By contrast, BAPTA-am exerted its effects via inhibition of HIF-prolyl hydroxylase activity and von Hippel-Lindau tumor repressor protein (VHL) interaction. Thus, calcium appeared to have a critical role in the regulation of the HIF system and subsequent activation of the PAI-1 gene expression. (Blood. 2004;104:3993-4001)
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Cui, Xiao-Lan, Wen-Wu Jin, Ya-Xian Ding, Larry D. Alexander, Ulrich Hopfer, and Janice G. Douglas. "Ca2+-dependent activation of c-junNH2-terminal kinase in primary rabbit proximal tubule epithelial cells." American Journal of Physiology-Cell Physiology 279, no. 2 (August 1, 2000): C403—C409. http://dx.doi.org/10.1152/ajpcell.2000.279.2.c403.
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Previous work from this laboratory demonstrated that arachidonic acid activates c- junNH2-terminal kinase (JNK) through oxidative intermediates in a Ca2+-independent manner (Cui X and Douglas JG. Arachidonic acid activates c- jun N-terminal kinase through NADPH oxidase in rabbit proximal tubular epithelial cells. Proc Natl Acad Sci USA 94: 3771–3776, 1997.). We now report that JNK can also be activated via a Ca2+-dependent mechanism by agents that increase the cytosolic Ca2+concentration (Ca2+ionophore A23187, Ca2+-ATPase inhibitor thapsigargin) or deplete intracellular Ca2+stores [intracellular Ca2+chelator 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid (BAPTA)-AM]. The activation of JNK by BAPTA-AM occurs despite a decrease in cytosolic Ca2+concentration as detected by the indicator dye fura 2, but appears to be related to Ca2+metabolism, because modification of BAPTA with two methyl groups increases not only the chelation affinity for Ca2+, but also the potency for JNK activation. BAPTA-AM stimulates Ca2+influx across the plasma membrane, and the resulting local Ca2+increases are probably involved in activation of JNK because Ca2+influx inhibitors (SKF-96365, nifedipine) and lowering of the free extracellular Ca2+concentration with EGTA reduce the BAPTA-induced JNK activation.
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Tymianski, M., M. P. Charlton, P. L. Carlen, and C. H. Tator. "Properties of neuroprotective cell-permeant Ca2+ chelators: effects on [Ca2+]i and glutamate neurotoxicity in vitro." Journal of Neurophysiology 72, no. 4 (October 1, 1994): 1973–92. http://dx.doi.org/10.1152/jn.1994.72.4.1973.
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1. Cell-permeant Ca2+ chelators such as 1,2-bis-(2-amino-phenoxy)ethane- N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) protect neurons against excitotoxic and ischemic neuronal injury in vitro and in vivo. Here we provide the first steps toward characterizing the mechanisms by which these agents produce their neuroprotective effects. 2. Cultured mouse spinal neurons were simultaneously loaded with the Ca2+ indicator fura-2 and with one of three permeant chelators derived from the fast Ca2+ buffer BAPTA, or with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid acetoxymethyl ester (EGTA-AM). Adding these chelators did not interfere with the fluorescence spectrum of fura-2 and had no effect on baseline [Ca2+]i. 3. The neurons were challenged with 250 microM L-glutamate for 50 min, producing a marked transient [Ca2+]i increase followed by a decay of [Ca2+]i to a lower “plateau.” About 80% of control neurons succumbed to this excitotoxic insult. Neurons that survived adjusted their plateau [Ca2+]i to lower levels than those that succumbed. 4. Neurons that were pretreated with permeant Ca2+ chelators became more resistant to these neurotoxic challenges. 5. We examined whether this reduction in glutamate neurotoxicity could be related to the given buffer's known Ca2+ affinity (Kd), its Ca2+ binding kinetics, and its ability to attenuate glutamate-induced [Ca2+]i increases. 6. Pretreatment of neurons with BAPTA analogues having Kds ranging from 100 to 3,600 microM 1) attenuated the amplitude and 2) lengthened the time constant describing the rise and decay of the glutamate-evoked [Ca2+]i transient. The magnitude of these effects paralleled the affinity of the chelator for Ca2+. 7. BAPTA-AM and its analogues dramatically attenuated the early neurotoxicity of glutamate, reducing cell deaths by up to 80%. However, in contrast with the graded effects of chelators having different Ca2+ affinities on Ca2+ transients, all BAPTA analogues were equally protective. These protective effects did not relate to the chelators' Ca2+ affinity within a Kd range of 100 nM (for BAPTA) to 3,600 nM (for 5,5'-dibromo BAPTA). 8. BAPTA-AM protected neurons in a concentration-dependent manner with 50% protection obtained with 10 microM, a concentration having no effect on the [Ca2+]i transient amplitude. 9. EGTA, a slow Ca2+ buffer with a similar Ca2+ affinity to BAPTA produced the same effects as BAPTA on [Ca2+]i transient kinetics. However, it was far less protective than BAPTA. 10. The time course of early glutamate neurotoxicity was altered by the BAPTA analogues, but not EGTA. BAPTA analogues caused a small increase in cell deaths in the first minutes of each experiment, followed by relative sparing from further neurodegeneration. 11. The ability of low Ca2+ affinity chelators such as 5,5'-dibromo BAPTA to protect neurons without markedly attenuating measured [Ca2+]i increases conflicts with the hypothesis that global elevations in [Ca2+]i are responsible for triggering neurotoxicity.(ABSTRACT TRUNCATED AT 400 WORDS)
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Maloney, Judith A., Oxana M. Tsygankova, Lijun Yang, Qiuyang Li, Agnieszka Szot, Kemal Baysal, and John R. Williamson. "Activation of ERK by Ca2+store depletion in rat liver epithelial cells." American Journal of Physiology-Cell Physiology 276, no. 1 (January 1, 1999): C221—C230. http://dx.doi.org/10.1152/ajpcell.1999.276.1.c221.
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In rat liver epithelial (WB) cells, Ca2+ pool depletion induced by two independent methods resulted in activation of extracellular signal-regulated protein kinase (ERK). In the first method, Ca2+ pool depletion by thapsigargin increased the activity of ERK, even when rise in cytosolic Ca2+ was blocked with the Ca2+ chelator BAPTA-AM. For the second method, addition of extracellular EGTA at a concentration shown to deplete intracellular Ca2+pools also increased ERK activity. In each instance, ERK activation, as measured by an immunocomplex kinase assay, was greatly reduced by the tyrosine kinase inhibitor genistein, suggesting that Ca2+ store depletion increased ERK activity through a tyrosine kinase pathway. The intracellular Ca2+-releasing agent thapsigargin increased Fyn activity, which was unaffected by BAPTA-AM pretreatment, suggesting that Fyn activity was unaffected by increased cytosolic free Ca2+. Furthermore, depletion of intracellular Ca2+ with EGTA caused inactivation of protein phosphatase 2A and protein tyrosine phosphatases. ANG II-induced activations of Fyn, Raf-1, and ERK were augmented in cells pretreated with BAPTA-AM, but ANG II-induced expression of the dual-specificity phosphatase mitogen-activated protein kinase phosphatase-1 was blocked by BAPTA-AM pretreatment. Together these results indicate that ERK activity is regulated by the balance of phosphorylation vs. dephosphorylation reactions in intact cells and that the amount of Ca2+stored in intracellular pools plays an important role in this regulation.
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Horváth, Balázs, Bence Hegyi, Kornél Kistamás, Krisztina Váczi, Tamás Bányász, János Magyar, Norbert Szentandrássy, and Péter P. Nánási. "Cytosolic calcium changes affect the incidence of early afterdepolarizations in canine ventricular myocytes." Canadian Journal of Physiology and Pharmacology 93, no. 7 (July 2015): 527–34. http://dx.doi.org/10.1139/cjpp-2014-0511.
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This study was designed to investigate the influence of cytosolic Ca2+ levels ([Ca2+]i) on action potential duration (APD) and on the incidence of early afterdepolarizations (EADs) in canine ventricular cardiomyocytes. Action potentials (AP) of isolated cells were recorded using conventional sharp microelectrodes, and the concomitant [Ca2+]i was monitored with the fluorescent dye Fura-2. EADs were evoked at a 0.2 Hz pacing rate by inhibiting the rapid delayed rectifier K+ current with dofetilide, by activating the late sodium current with veratridine, or by activating the L-type calcium current with BAY K8644. These interventions progressively prolonged the AP and resulted in initiation of EADs. Reducing [Ca2+]i by application of the cell-permeant Ca2+ chelator BAPTA-AM lengthened the AP at 1.0 Hz if it was applied alone, in the presence of veratridine, or in the presence of BAY K8644. However, BAPTA-AM shortened the AP if the cells were pretreated with dofetilide. The incidence of the evoked EADs was strongly reduced by BAPTA-AM in dofetilide, moderately reduced in veratridine, whereas EAD incidence was increased by BAPTA-AM in the presence of BAY K8644. Based on these experimental data, changes in [Ca2+]i have marked effects on APD as well as on the incidence of EADs; however, the underlying mechanisms may be different, depending on the mechanism of EAD generation. As a consequence, reduction of [Ca2+]i may eliminate EADs under some, but not all, experimental conditions.
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Miteva, Anna, Alexander Gaydukov, and Olga Balezina. "Interaction between Calcium Chelators and the Activity of P2X7 Receptors in Mouse Motor Synapses." International Journal of Molecular Sciences 21, no. 6 (March 16, 2020): 2034. http://dx.doi.org/10.3390/ijms21062034.
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The ability of P2X7 receptors to potentiate rhythmically evoked acetylcholine (ACh) release through Ca2+ entry via P2X7 receptors and via L-type voltage-dependent Ca2+ channels (VDCCs) was compared by loading Ca2+ chelators into motor nerve terminals. Neuromuscular preparations of the diaphragms of wild-type (WT) mice and pannexin-1 knockout (Panx1−/−) mice, in which ACh release is potentiated by the disinhibition of the L-type VDCCs upon the activation of P2X7 receptors, were used. Miniature end-plate potentials (MEPPs) and evoked end-plate potentials (EPPs) were recorded when the motor terminals were loaded with slow or fast Ca2+ chelators (EGTA-AM or BAPTA-AM, respectively, 50 μM). In WT and Panx1−/− mice, EGTA-AM did not change either spontaneous or evoked ACh release, while BAPTA-AM inhibited synaptic transmission by suppressing the quantal content of EPPs throughout the course of the short rhythmic train (50 Hz, 1 s). In the motor synapses of either WT or Panx1−/− mice in the presence of BAPTA-AM, the activation of P2X7 receptors by BzATP (30 μM) returned the EPP quantal content to the control level. In the neuromuscular junctions (NMJs) of Panx1−/− mice, EGTA-AM completely prevented the BzATP-induced increase in EPP quantal content. After Panx1−/− NMJs were treated with BAPTA-AM, BzATP lost its ability to enhance the EPP quantal content to above the control level. Nitrendipine (1 μM), an inhibitor of L-type VDCCs, was unable to prevent this BzATP-induced enhancement of EPP quantal content to the control level. We propose that the activation of P2X7 receptors may provide additional Ca2+ entry into motor nerve terminals, which, independent of the modulation of L-type VDCC activity, can partially reduce the buffering capacity of Ca2+ chelators, thereby providing sufficient Ca2+ signals for ACh secretion at the control level. However, the activity of both Ca2+ chelators was sufficient to eliminate Ca2+ entry via L-type VDCCs activated by P2X7 receptors and increase the EPP quantal content in the NMJs of Panx1−/− mice to above the control level.
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Sun, Y. B., C. Caputo, and K. A. P. Edman. "Effects of BAPTA on force and Ca2+ transient during isometric contraction of frog muscle fibers." American Journal of Physiology-Cell Physiology 275, no. 2 (August 1, 1998): C375—C381. http://dx.doi.org/10.1152/ajpcell.1998.275.2.c375.
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The effects of 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid (BAPTA) on force and intracellular Ca2+ transient were studied during isometric twitches and tetanuses in single frog muscle fibers. BAPTA was added to the bathing solution in its permeant AM form (50 and 100 μM). There was no clear correlation between the changes in force and the changes in Ca2+ transient. Thus during twitch stimulation BAPTA did not suppress the Ca2+ transient until the force had been reduced to <50% of its control value. At the same time, the peak myoplasmic free Ca2+concentration reached during tetanic stimulation was markedly increased, whereas the force was slightly reduced by BAPTA. The effects of BAPTA were not duplicated by using another Ca2+ chelator, EGTA, indicating that BAPTA may act differently as a Ca2+ chelator. Stiffness measurements suggest that the decrease in mechanical performance in the presence of BAPTA is attributable to a reduced number of active cross bridges. The results could mean that BAPTA, under the conditions used, inhibits the binding of Ca2+ to troponin C resulting in a reduced state of activation of the contractile system.
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AOYAMA, Masako, Dale R. GRABOWSKI, George R. DUBYAK, Andreas I. CONSTANTINOU, Lisa A. RYBICKI, Ronald M. BUKOWSKI, Mahrukh K. GANAPATHI, Ian D. HICKSON, and Ram GANAPATHI. "Attenuation of drug-stimulated topoisomerase II–DNA cleavable complex formation in wild-type HL-60 cells treated with an intracellular calcium buffer is correlated with decreased cytotoxicity and site-specific hypophosphorylation of topoisomerase IIα." Biochemical Journal 336, no. 3 (December 15, 1998): 727–33. http://dx.doi.org/10.1042/bj3360727.
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Topoisomerase II (topo II), an essential enzyme for cell viability, is also the target for clinically important anti-neoplastic agents that stimulate topo II-mediated DNA scission. The role of alterations in topo IIα phosphorylation and its effect on drug-induced DNA damage and cytotoxicity were investigated. Following loading of HL-60 cells with the calcium buffer 1,2-bis-(o-aminophenoxy)ethane-N,N,N´,N´-tetra-acetic acid tetra(acetoxymethyl) ester (BAPTA-AM), which abrogates intracellular Ca2+ transients, a significant decrease in etoposide (VP-16)- or amsacrine (m-AMSA)-stabilized topo II–DNA cleavable complex formation and a corresponding decrease in cytotoxicity was observed. In a cell-free system, nuclear extracts from BAPTA-AM-treated cells exhibited markedly less activity when assayed for VP-16-stabilized topo II–DNA complex formation, but not decatenation of kinetoplast DNA. In contrast, the loading of HL-60 cells with N,N,N´,N´-tetrakis-(2-pyridyl)ethylenediamine (TPEN), which binds heavy metals without disturbing calcium or magnesium concentrations, did not significantly affect VP-16-stimulated topo II–DNA cleavable complex formation or cytotoxicity. In HL-60 cells the accumulation of BAPTA, but not TPEN, also led to the hypophosphorylation of topo IIα. Tryptic phosphopeptide mapping of topo IIα protein from HL-60 cells revealed: (a) eight major phosphorylation sites in untreated cells; (b) hypophosphorylation of two out of eight sites in BAPTA-AM-treated cells; and (c) hypophosphorylation of between two and four out of eight sites in topo II-poison-resistant HL-60 cells. The two hypophosphorylated sites present following BAPTA-AM treatment of wild-type cells were identical with the hypophosphorylated sites in the resistant cells, but were not the same as the sites that are substrates for casein kinase II [Wells, Addison, Fry, Ganapathi and Hickson (1994) J. Biol. Chem. 269, 29746–29751]. In summary, changes in intracellular Ca2+ transients that lead to the site-specific hypophosphorylation of topo IIα are possibly involved in regulating the DNA damage caused by and the cytotoxic potential of topo II poisons.
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Tsukii, Katsuyoshi, Norimichi Nakahata, Susumu Tsurufuji, and Yasushi Ohizumi. "Ca2+-independent synergistic augmentation of O2- production by FMLP and PMA in HL-60 cells." Canadian Journal of Physiology and Pharmacology 76, no. 10-11 (October 1, 1998): 1024–32. http://dx.doi.org/10.1139/y98-089.
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N-Formyl-Met-Leu-Phe (FMLP) and phorbol 12-myristate 13-acetate (PMA) caused a synergistic augmentation of superoxide anion (O2-) production in neutrophil-like HL-60 cells differentiated with dibutyryl cAMP. The present study was undertaken to investigate the mechanism of the synergistic augmentation of O2- production. FMLP increased intracellular free Ca2+ concentration ([Ca2+]i), which was slightly suppressed by PMA and completely inhibited by an intracellular Ca2+ chelating agent, O,Oprime-bis(2-aminophenyl)ethyleneglycol-N,N,Nprime,Nprime-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM). Although FMLP-induced O2- production was inhibited by BAPTA-AM, a major part of the synergistic augmentation of O2- production by FMLP and PMA remained after BAPTA-AM treatment, suggesting that a Ca2+-independent mechanism might be involved in the augmentation. FMLP and PMA caused an activation of phospholipase D (PLD) almost additively in a Ca2+-sensitive manner. The synergistic activation of mitogen-activated protein kinase (MAPK) was evoked by combined addition of PMA and FMLP in a BAPTA-AM resistant manner. However, PD98059, a MAPK kinase inhibitor, did not affect the synergistic augmentation of O2- production, although it potently inhibited the synergistic augmentation of tyrosine phosphorylation of MAPK. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, inhibited FMLP-induced O2- production, but it did not inhibit the synergistic augmentation of O2- production by PMA and FMLP. In contrast, staurosporine and GF109203X, protein kinase C inhibitors, reduced the synergistic augmentation induced by PMA and FMLP. In addition, pertussis toxin (PT) abolished the synergistic augmentation of O2- production. It is concluded that the synergistic augmentation of O2- production induced by PMA and FMLP is mediated through a PT-sensitive G protein and a protein kinase C in a Ca2+-independent manner.Key words: superoxide anion, phorbol 12-myristate 13-acetate, N-formyl-Met-Leu-Phe, intracellular Ca2+ ions ; protein kinase C.
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Zheng, Feng, Taijun Hang, Chunyong Wu, Bin Di, Wenyin Liu, Jihua Xu, Chunxiao Zhai, Di Sun, Jinxing Liu, and Wei Wei. "Determination of BAPTA-AM, the acetoxymethyl tetraester of BAPTA, in rat plasma by liquid chromatography tandem mass spectrometry." Journal of Mass Spectrometry 41, no. 12 (2006): 1615–22. http://dx.doi.org/10.1002/jms.1125.
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Gioino, Paula, Brendan G. Murray, and Juan P. Ianowski. "Serotonin triggers cAMP and PKA-mediated intracellular calcium waves in Malpighian tubules of Rhodnius prolixus." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 307, no. 7 (October 1, 2014): R828—R836. http://dx.doi.org/10.1152/ajpregu.00561.2013.
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Rhodnius prolixus is a hematophagous insect vector of Chagas disease capable of ingesting up to 10 times its unfed body weight in blood in a single meal. The excess water and ions ingested with the meal are expelled through a rapid postprandial diuresis driven by the Malpighian tubules. Diuresis is triggered by at least two diuretic hormones, a CRF-related peptide and serotonin, which were traditionally believed to trigger cAMP as an intracellular second messenger. Recently, calcium has been suggested to act as a second messenger in serotonin-stimulated Malpighian tubules. Thus, we tested the role of calcium in serotonin-stimulated Malpighian tubules from R. prolixus. Our results show that serotonin triggers cAMP-mediated intracellular Ca2+ waves that were blocked by incubation in Ca2+-free saline containing the cell membrane-permeant Ca2+ chelator BAPTA-AM, or the PKA blocker H-89. Treatment with 8-Br-cAMP triggered Ca2+ waves that were blocked by H-89 and BAPTA-AM. Analysis of the secreted fluid in BAPTA-AM-treated tubules showed a 75% reduction in fluid secretion rate with increased K+ concentration, reduced Na+ concentration. Taken together, the results indicate that serotonin triggers cAMP and PKA-mediated Ca2+ waves that are required for maximal ion transport rate.
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Su, Xing-Li, Ying-Guang Liu, Man Shi, Yan-Yan Zhao, Xiang-Yan Liang, Li-Jun Zhang, Lan-Lan Wei, and Yu-Feng Zhao. "The GPR120 Agonist TUG-891 Inhibits the Motility and Phagocytosis of Mouse Alveolar Macrophages." BioMed Research International 2020 (February 22, 2020): 1–10. http://dx.doi.org/10.1155/2020/1706168.
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Movement and phagocytosis characterize the fundamental actions of macrophages. Although it is known that the free fatty acid receptor GPR120 is expressed in macrophages and regulates cytokine expression to exert anti-inflammatory activities, the effects of GPR120 activation on the motility and phagocytosis of macrophages are not clear. In this study, mouse alveolar macrophages (AM) were stimulated with the GPR120 agonist TUG-891, and the changes in cell motility, intracellular Ca2+ concentration ([Ca2+]i), and the ability of phagocytosis were measured. Mouse AM in controls exhibited active movement in vitro, and TUG-891 significantly restrained AM movement. Meanwhile, TUG-891 stimulated a quick increase in [Ca2+]i in AM, which was blocked separately by the Gq protein inhibitor YM-254890, the phospholipase C (PLC) inhibitor U73122, or depletion of endoplasmic reticulum (ER) Ca2+ store by thapsigargin. The inhibition of AM movement by TUG-891 was eliminated by YM-254890, U73122, thapsigargin, and chelation of cytosolic Ca2+ by BAPTA. Moreover, TUG-891 inhibited AM phagocytosis of fluorescent microspheres, which was also blocked by YM-254890, U73122, thapsigargin, and BAPTA. In conclusion, GPR120 activation in mouse AM increases [Ca2+]i but inhibits the motility and phagocytosis via Gq protein/PLC-mediated Ca2+ release from ER Ca2+ store.
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JORNOT, Lan, Hilke PETERSEN, and Alain F. JUNOD. "Hydrogen peroxide-induced DNA damage is independent of nuclear calcium but dependent on redox-active ions." Biochemical Journal 335, no. 1 (October 1, 1998): 85–94. http://dx.doi.org/10.1042/bj3350085.
Full textAbstract:
In cells undergoing oxidative stress, DNA damage may result from attack by •OH radicals produced by the Fenton reaction, and/or by nucleases activated by nuclear calcium. In the present study, the participation of these two mechanisms was investigated in HeLa cells. Nuclear-targeted aequorin was used for selectively monitoring Ca2+ concentrations within the nuclei ([Ca2+]n), in conjunction with the cell-permeant calcium chelator bis-(o-aminophenoxy)ethane-N,N,N´,N´-tetraacetic acid acetoxymethyl ester (BAPTA/AM), the lipid-soluble broad-spectrum metal chelator with low affinity for Ca2+ and Mg2+ N,N,N´,N´-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), and the high-affinity iron/copper chelator 1,10-phenanthroline (PHE). In Ca2+-containing medium, H2O2 induced extensive DNA strand breaks and an increase in [Ca2+]n that was almost identical to that observed in the cytosol ([Ca2+]c). In cells bathed in Ca2+-free/EGTA medium, in which the increases in [Ca2+]n and [Ca2+]c due to H2O2 were significantly reduced, similar levels of DNA fragmentation also occurred. In cells preloaded with BAPTA/AM or TPEN, the small increase of [Ca2+]n normally elicited by H2O2 in Ca2+-free medium was completely buffered, and DNA damage was largely prevented. On the other hand, pretreatment with PHE did not affect the calcium response in the nuclei, but completely prevented DNA strand breakage induced by H2O2. Re-addition of 100 µM CuSO4 and 100 µM FeSO4 to TPEN- and PHE-treated cells prior to H2O2 challenge reversed the effect of TPEN and PHE, whereas 1 mM was necessary to negate the effect of BAPTA/AM. The levels of DNA strand breakage observed, however, did not correlate with the amounts of 8-hydroxy 2´-deoxyguanosine (8-OHdG): H2O2 did not produce 8-OHdG, whereas PHE alone slightly increased 8-OHdG levels. CuSO4 and FeSO4 enhanced the effects of PHE, particularly in the presence of H2O2. Exposure of cells to a mixture of CuSO4/FeSO4 also resulted in a significant increase in 8-OHdG levels, which was prevented in cells preloaded with BAPTA/AM. Similar results were obtained in a cell-free system using isolated calf thymus DNA exposed to CuSO4/FeSO4, regardless of whether H2O2 was present or not. These results suggest that BAPTA/AM prevents H2O2-induced DNA damage by acting as an iron/copper chelator. These data also indicate that caution must be exercised in using Ca2+ chelating agents as evidence for a role in cellular Ca2+ levels in experimental conditions in which transition-metal-ion-mediated oxidant production is also occurring.
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Leganés, Francisco, Karl Forchhammer, and Francisca Fernández-Piñas. "Role of calcium in acclimation of the cyanobacterium Synechococcus elongatus PCC 7942 to nitrogen starvation." Microbiology 155, no. 1 (January 1, 2009): 25–34. http://dx.doi.org/10.1099/mic.0.022251-0.
Full textAbstract:
A Ca2+ signal is required for the process of heterocyst differentiation in the filamentous diazotrophic cyanobacterium Anabaena sp. PCC 7120. This paper presents evidence that a transient increase in intracellular free Ca2+ is also involved in acclimation to nitrogen starvation in the unicellular non-diazotrophic cyanobacterium Synechococcus elongatus PCC 7942. The Ca2+ transient was triggered in response to nitrogen step-down or the addition of 2-oxoglutarate (2-OG), or its analogues 2,2-difluoropentanedioic acid (DFPA) and 2-methylenepentanedioic acid (2-MPA), to cells growing with combined nitrogen, suggesting that an increase in intracellular 2-OG levels precedes the Ca2+ transient. The signalling protein PII and the transcriptional regulator NtcA appear to be needed to trigger the signal. Suppression of the Ca2+ transient by the intracellular Ca2+ chelator N,N′-[1,2-ethanediylbis(oxy-2,1-phenylene)]bis[N-[2-[(acetyloxy)methoxy]-2-oxoethyl]]-,bis[(acetyloxy)methyl] ester (BAPTA-AM) inhibited expression of the glnB and glnN genes, which are involved in acclimation to nitrogen starvation and transcriptionally activated by NtcA. BAPTA-AM treatment partially inhibited expression of the nblA gene, which is involved in phycobiliprotein degradation following nutrient starvation and is regulated by NtcA and NblR; in close agreement, BAPTA-AM treatment partially inhibited bleaching following nitrogen starvation. Taken together, the results presented here strongly suggest an involvement of a defined Ca2+ transient in acclimation of S. elongatus to nitrogen starvation through NtcA-dependent regulation.
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Negulescu, P. A., W. W. Reenstra, and T. E. Machen. "Intracellular Ca requirements for stimulus-secretion coupling in parietal cell." American Journal of Physiology-Cell Physiology 256, no. 2 (February 1, 1989): C241—C251. http://dx.doi.org/10.1152/ajpcell.1989.256.2.c241.
Full textAbstract:
The role of cytosolic free Ca (Cai) in stimulating acid production by the parietal cell in response to the secretagogues carbachol (carb), histamine (hist), and dibutyryl adenosine 3',5'-cyclic monophosphate plus 3-isobutyl-1-methylxanthine (DBcAMP + IBMX) was evaluated. Microfluorimetry with fura-2 was used to measure Cai in single parietal cells within intact rabbit gastric glands. Acid production was determined in parallel experiments by monitoring the accumulation of [14C]aminopyrine (AP) in suspensions of glands. Carb increased peak Cai levels to 1 microM in a dose-dependent manner [concentration for half-maximal response (K0.5) = 8 microM] that correlated well with the dose dependence of carb-stimulated AP accumulation (K0.5 = 18 microM). The Ca chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was used to attenuate secretagogue-induced Cai increases. Incubating glands for 10 min with 1 and 10 microM BAPTA/AM caused resting Cai to decrease from 119 to 93 and 80 nM, respectively. BAPTA/AM, 1 and 10 microM, blocked carb-stimulated increases in Cai by 60 and 90% and AP accumulation by 50 and 90%. Hist, which increases cytosolic cAMP, caused small and relatively infrequent increases in Cai. Even so, hist-stimulated AP accumulation was inhibited 8 and 40% by 1 and 10 microM BAPTA. DBcAMP had no effect on Cai, and AP accumulation caused by DBcAMP was unaffected by the concentrations of BAPTA tested. These data suggest that carb requires an increase in Cai as a secretory signal. Hist also exhibited some Cai dependence, which may be attributable to either a small increase in Cai or the necessity of having a specific basal level of Cai. A Cai requirement for DBcAMP-stimulated acid secretion was not detected. Thus the parietal cell possesses both Ca-dependent and Ca-independent stimulatory pathways, and at least one secretagogue (hist) may utilize both pathways.
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Song, S. K., R. S. Hotchkiss, J. Neil, P. E. Morris, C. Y. Hsu, and J. J. Ackerman. "Determination of intracellular calcium in vivo via fluorine-19 nuclear magnetic resonance spectroscopy." American Journal of Physiology-Cell Physiology 269, no. 2 (August 1, 1995): C318—C322. http://dx.doi.org/10.1152/ajpcell.1995.269.2.c318.
Full textAbstract:
Fluorine-19-nuclear magnetic resonance (19F-NMR) spectroscopic detection of the NMR-active Ca2+ indicator 5-fluoro-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (5F-BAPTA) is one method for measuring cytosolic free Ca2+ concentration ([Ca2+]i) and has been used previously to measure [Ca2+]i in isolated cells and perfused organs. The aim of the present investigation was to demonstrate the feasibility of determining [Ca2+]i in vivo and in situ using 19F-NMR and 5F-BAPTA. Experiments were performed on male Sprague-Dawley rats with a surface-coil antenna employed for NMR interrogation. The Ca2+ indicator, 5F-BAPTA, was infused either intravenously (kidney, spleen) or intraventricularly (brain) as a 100 mg/ml solution of the cell-permeant acetoxymethyl ester (5F-BAPTA-AM) in dimethyl sulfoxide. Rats tolerated intravenous infusion without evident change in mean arterial blood pressure. In all tissues examined, kidney, spleen, and brain, [Ca2+]i was approximately 200 nM. To our knowledge, these results represent the first in vivo and in situ determinations of [Ca2+]i employing 19F-NMR.
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HIRANO, Daisuke, Yoshiko AOKI, Hiroyuki OGASAWARA, Hisashi KODAMA, Iwao WAGA, Chie SAKANAKA, Takao SHIMIZU, and Motonao NAKAMURA. "Functional coupling of adenosine A2a receptor to inhibition of the mitogen-activated protein kinase cascade in Chinese hamster ovary cells." Biochemical Journal 316, no. 1 (May 15, 1996): 81–86. http://dx.doi.org/10.1042/bj3160081.
Full textAbstract:
Activation of Gs-coupled receptors enhances the increase in cyclic AMP mediated by adenylate cyclases. As it has been shown that cyclic AMP inhibits the epidermal growth factor-activated mitogen-activated protein kinase (MAPK) signalling pathway, stimulation of Gs-coupled receptors may lead to the inhibition of MAPK activation. To investigate the effect of a Gs-coupled receptor on the MAPK cascade, we cloned the adenosine (Ado) A2a receptor from a guinea-pig leucocyte cDNA library, and established Chinese hamster ovary (CHO) cells stably expressing the receptor (CHOAdoA2R). The [3H]5´-N-ethylcarbamoyladenosine (NECA) binding characteristics (Kd = 91.0±5.4 nM, Bmax = 707±11 fmol/mg of protein, n = 3) and NECA-induced cyclic AMP production indicate that the cloned Ado A2a receptor was functionally expressed in the cells. In CHO cells, thrombin induced intracellular Ca2+ increase and MAPK activation through the intrinsic G-coupled receptor. In CHOAdoA2R cells, NECA partially inhibited thrombin-elicited MAPK activation. When combining NECA-treatment with 1,2-bis-(o-aminophenoxy)ethane-N,N,N´,N´-tetra-acetic acid acetoxymethyl ester (BAPTA-AM) loading, a nearly complete inhibition of the MAPK activation occurred. Forskolin also partially inhibited the MAPK activation and synergized with BAPTA-AM, suggesting that partial inhibition of MAPK activation by NECA results from cyclic AMP production via Ado A2a receptor activation. The same synergism of MAPK inhibition between wortmannin and BAPTA-AM was observed, but not between wortmannin and NECA. These results suggest that cyclic AMP production through Ado A2a receptor inhibits thrombin-elicited MAPK activation by a Ca2+-independent/ wortmannin-sensitive pathway in CHO cells.
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Okolo, Charles, Thomas Wong, Mark W. Moody, and Toan D. Nguyen. "Effects of bile acids on dog pancreatic duct epithelial cell secretion and monolayer resistance." American Journal of Physiology-Gastrointestinal and Liver Physiology 283, no. 5 (November 1, 2002): G1042—G1050. http://dx.doi.org/10.1152/ajpgi.00436.2001.
Full textAbstract:
Pancreatic duct epithelial cells (PDEC) mediate the secretion of fluid and electrolytes and are exposed to refluxed bile. In nontransformed cultured dog PDEC, which express many ion transport pathways of PDEC, 1 mM taurodeoxycholic acid (TDCA) stimulated an125I−efflux inhibited by DIDS and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and a86Rb+efflux inhibited by charybdotoxin. Inhibition by 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid (BAPTA)-AM suggests mediation via increased intracellular Ca2+concentration, whereas the absence of lactate dehydrogenase release excludes cellular toxicity. At 1 mM, TDCA stimulated a larger125I−efflux than glycodeoxycholate; two dihydroxy bile acids, taurochenodeoxycholate and TDCA, were similarly effective, whereas a trihydroxy bile acid, taurocholate, was ineffective. In Ussing chambers, 1 mM serosal or 2 mM luminal TDCA stimulated an Iscincrease from confluent PDEC monolayers. TDCA also stimulated 1) a short-circuit current ( Isc) increase from basolaterally permeabilized PDEC subject to a serosal-to-luminal Cl−gradient that was inhibited by BAPTA-AM, DIDS, and NPPB and 2) an Iscincrease from apically permeabilized PDEC subject to a luminal-to-serosal K+gradient inhibited by BAPTA-AM and charybdotoxin. Along with the efflux studies, these findings suggest that TDCA interacts directly with PDEC to stimulate Ca2+-activated apical Cl−channels and basolateral K+channels. Monolayer transepithelial resistance was only minimally affected by 1 mM serosal and 2 mM luminal TDCA but decreased after exposure to higher TDCA concentrations (2 mM serosal and 4 mM luminal). A secretory role for bile acids should be considered in pancreatic diseases associated with bile reflux.
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Schubert, M. L., and J. Hightower. "Functionally distinct muscarinic receptors on gastric somatostatin cells." American Journal of Physiology-Gastrointestinal and Liver Physiology 258, no. 6 (June 1, 1990): G982—G987. http://dx.doi.org/10.1152/ajpgi.1990.258.6.g982.
Full textAbstract:
The present study was designed to examine the mode of action of muscarinic agonists on somatostatin secretion in intact gastric tissues, i.e., mucosal segments from the fundus and antrum of rat and the isolated luminally perfused mouse stomach. Methacholine caused similar decreases in somatostatin secretion in segments from the fundus (35 +/- 3%; P less than 0.001) and antrum (35 +/- 2%; P less than 0.001) of rat stomach, and in whole mouse stomach (43 +/- 3%; P less than 0.001). The decrease was the net effect of a dominant inhibition and a lesser stimulation of somatostatin secretion. Pretreatment with the permeant derivative of the acetomethoxy ester form of the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA/AM, 15 microM) caused a further decrease in methacholine-induced somatostatin secretion, implying that a stimulatory component existed that was mediated by intracellular calcium. Pretreatment with pertussis toxin (125 ng/ml) for 60 min converted the decrease in somatostatin secretion to an increase above basal levels. The increase induced by pretreatment with pertussis toxin was abolished by additional pretreatment with BAPTA/AM. Procaine (5 mM), which blocks release of calcium from intracellular stores, produced an effect on somatostatin secretion similar to that of BAPTA/AM. The results indicate that 1) methacholine exerts dual inhibitory and stimulatory effects on somatostatin cells of rat and mouse stomach, 2) the dominant effect is inhibitory and sensitive to pertussis toxin, and 3) a concurrent stimulatory effect, mediated by calcium, is unmasked after blockade of the inhibitory effect with pertussis toxin.
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Merriam, Laura A., Fabiana S. Scornik, and Rodney L. Parsons. "Ca2+-Induced Ca2+ Release Activates Spontaneous Miniature Outward Currents (SMOCs) in Parasympathetic Cardiac Neurons." Journal of Neurophysiology 82, no. 2 (August 1, 1999): 540–50. http://dx.doi.org/10.1152/jn.1999.82.2.540.
Full textAbstract:
Mudpuppy parasympathetic cardiac neurons exhibit spontaneous miniature outward currents (SMOCs) that are thought to be due to the activation of clusters of large conductance Ca2+-activated K+ channels (BK channels) by localized release of Ca2+ from internal stores close to the plasma membrane. Perforated-patch whole cell recordings were used to determine whether Ca2+-induced Ca2+ release (CICR) is involved in SMOC generation. We confirmed that BK channels are involved by showing that SMOCs are inhibited by 100 nM iberiotoxin or 500 μM tetraethylammonium (TEA), but not by 100 nM apamin. SMOC frequency is decreased in solutions that contain 0 Ca2+/3.6 mM Mg2+, and also in the presence of 1 μM nifedipine and 3 μM ω-conotoxin GVIA, suggesting that SMOC activation is dependent on calcium influx. However, Ca2+ influx alone is not sufficient; SMOC activation is also dependent on Ca2+release from the caffeine- and ryanodine-sensitive Ca2+store, because exposure to 2 mM caffeine consistently caused an increase in SMOC frequency, and 10–100 μM ryanodine altered the configuration of SMOCs and eventually inhibited SMOC activity. Depletion of intracellular Ca2+ stores by the Ca-ATPase inhibitor cyclopiazonic acid (10 μM) inhibited SMOC activity, even when Ca2+ influx was not compromised. We also tested the effects of the membrane-permeable Ca2+ chelators, bis-( o-aminophenoxy)- N,N,N′,N′-tetraacetic acid-AM (BAPTA-AM) and EGTA-AM. EGTA-AM (10 μM) caused no inhibition of SMOC activation, whereas 10 μM BAPTA-AM consistently inhibited SMOCs. After SMOCs were completely inhibited by BAPTA, 3 mM caffeine caused SMOC activity to resume. This effect was reversible on removal of caffeine and suggests that the source of Ca2+ that triggers the internal Ca2+ release channel is different from the source of Ca2+ that activates clusters of BK channels. We propose that influx of Ca2+ through voltage-dependent Ca2+ channels is required for SMOC generation, but that the influx of Ca2+ triggers CICR from intracellular stores, which then activates the BK channels responsible for SMOC generation.
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Morris, Philip E. "SYNTHESIS OF 5,5′DIFLUORO-BAPTA-AM, A USEFULIN VIVOPROBE FOR CALCIUM DETERMINATION." Organic Preparations and Procedures International 25, no. 4 (August 1993): 445–48. http://dx.doi.org/10.1080/00304949309457987.
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McLennan, H. J., M. L. Sutton-McDowall, S. Heng, and J. G. Thompson. "153 Fertilisation of Cattle Oocytes is Linked to Novel Waves of a Ca2+ Fluorophore in Cumulus Cells." Reproduction, Fertility and Development 30, no. 1 (2018): 216. http://dx.doi.org/10.1071/rdv30n1ab153.
Full textAbstract:
During fertilization, multiple intracellular calcium (Ca2+) oscillations are initiated after sperm binding to the oocyte vitelline membrane. This Ca2+ signalling has been extensively studied in denuded mouse and Xenopus oocytes but minimally studied in larger mammals. Cows in particular are unusual, as the few studies on oocyte activation have observed fewer Ca2+ oscillations during fertilisation compared with mice. Furthermore, cattle intracytoplasmic sperm injection (ICSI) is inefficient, despite parthenogenetic activation occurring readily. We hypothesise that cumulus cells are important for cattle oocyte activation at fertilisation. Here, we assessed the behaviour of Ca2+oscillations in fertilising intact cattle cumulus–oocyte complexes (COC). Abattoir-derived cattle COC were matured and fertilised in vitro using Bovine Vitro Media Suite (IVF Vet Solutions). The COC were stained 3.5 h after insemination with the Ca2+ fluorescent probe Fluo4AM (5 μM, Molecular Probes Inc., Eugene, OR, USA) for 30 min, washed, and imaged every 5 min for 6 h in a Fluoview FV10i incubating time-lapse confocal microscope (Olympus) before being returned to culture. Embryo development was assessed at Day 8 to confirm fertilisation. Fluo4AM fluorescence intensity was assessed using FIJI ImageJ. Mean relative intensity over time was graphed for specific regions of interest and the area under graphs was calculated to quantify differences for comparison using a Mann-Whitney Test (mean ± SEM). Experiment 1 (4 reps of 10 COC) compared confirmed fertilised v. uninseminated; experiment 2 (2 reps of 10 COC) compared inseminated COC ± 10 μM BAPTA-AM (Ca2+ chelator, Sigma-Aldrich, St. Louis, MO, USA). There were distinct coordinated waves of differing Fluo4AM intensity in both the oocyte and the cumulus cells surrounding the confirmed fertilised oocytes. This contrasted to the random uncoordinated flashes of Fluo4AM fluorescence in the cumulus cells of the uninseminated oocytes. The fluorescence pattern in +BAPTA-AM COC matched the random flashes observed in the uninseminated group of experiment 1. The fluorescence in the media surrounding the COC immediately following the Fluo4AM waves spiked and then plateaued at a higher level of fluorescence. This was quantified by assessing the area under the graph for 1 h of the plateau following the fluorescence spike. There were no differences between confirmed fertilised (346.4 ± 41.62) and uninseminated groups (239.8 ± 32.08; P > 0.05), but this was affected by differences in cumulus dispersal due to the presence or absence of sperm. Experiment 2 used BAPTA-AM to block oocyte activation with sperm present in both groups and showed a significant difference between the fluorescence increase in the media of the 2 groups (–BAPTA-AM: 311.2 ± 31.57, +BAPTA-AM: 201.4 ± 26.59; P < 0.03). Although the physiological significance has yet to be determined, we have observed a novel Ca2+ wave in the cumulus cells that could be linked to oocyte activation in cattle. There was a significant increase in Fluo4AM fluorescence in the media surrounding the COC, which may indicate cumulus cells are releasing Ca2+ at the time of oocyte activation.
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Yoo, Je-Ok, Chang-Hee Lee, Byeong-Moon Hwang, Woo Jin Kim, Young-Myeong Kim, and Kwon-Soo Ha. "Regulation of intracellular Ca2+ in the cytotoxic response to photodynamic therapy with a chlorin-based photosensitizer." Journal of Porphyrins and Phthalocyanines 13, no. 07 (July 2009): 811–17. http://dx.doi.org/10.1142/s1088424609001066.
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We investigated regulation of intracellular Ca2+ induced by photodynamic therapy (PDT) with a new chlorin-based photosensitizer, DH-II-24, in human gastric adenocarcinoma cells. DH-II-24-mediated PDT induced necrotic cell death according to post-irradiation time, and produced intracellular reactive oxygen species (ROS) in an irradiation time-dependent manner. PDT also increased intracellular Ca2+ , and this Ca2+ elevation was largely inhibited by BAPTA-AM but not by EGTA. BAPTA-AM inhibited the ROS production by PDT, whereas NAC and Trolox had no effect on the PDT-induced Ca2+ response. In the presence of EGTA, pre-incubation with thapsigargin, Gly-Phe-β-naphthylamide or brefeldin A had no significant effect on the PDT-induced elevation in intracellular Ca2+ . However, ruthenium red affected the initial and late Ca2+ responses to PDT. Thus, DH-II-24-mediated PDT produces intracellular ROS via elevation in intracellular Ca2+ , contributed, at least in part, by mitochondria, which results in necrotic death of the human gastric adenocarcinoma cells.
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Zhu, Yi, Li He, Jing Qu, and Yong Zhou. "Regulation of Vascular Smooth Muscle Cell Stiffness and Adhesion by [Ca2+]i: An Atomic Force Microscopy-Based Study." Microscopy and Microanalysis 24, no. 6 (December 2018): 708–12. http://dx.doi.org/10.1017/s1431927618015519.
Full textAbstract:
AbstractThe intracellular concentration of calcium ion ([Ca2+]i) is a critical regulator of cell signaling and contractility of vascular smooth muscle cells (VSMCs). In this study, we employed an atomic force microscopy (AFM) nanoindentation-based approach to investigate the role of [Ca2+]i in regulating the cortical elasticity of rat cremaster VSMCs and the ability of rat VSMCs to adhere to fibronectin (Fn) matrix. Elevation of [Ca2+]i by ionomycin treatment increased rat VSMC stiffness and cell adhesion to Fn-biofunctionalized AFM probes, whereas attenuation of [Ca2+]i by 1,2-Bis (2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM) treatment decreased the mechanical and matrix adhesive properties of VSMCs. Furthermore, we found that ionomycin/BAPTA-AM treatments altered expression of α5 integrin subunits and α smooth muscle actin in rat VSMCs. These data suggest that [Ca2+]i regulates VSMC elasticity and adhesion to the extracellular matrix by a potential mechanism involving changing dynamics of the integrin–actin cytoskeleton axis.
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Li, Jin, Jihong Qu, and Richard D. Nathan. "Ionic basis of ryanodine’s negative chronotropic effect on pacemaker cells isolated from the sinoatrial node." American Journal of Physiology-Heart and Circulatory Physiology 273, no. 5 (November 1, 1997): H2481—H2489. http://dx.doi.org/10.1152/ajpheart.1997.273.5.h2481.
Full textAbstract:
Spontaneous electrical activity and indo 1 fluorescence ratios were recorded simultaneously in cultured pacemaker cells isolated from the rabbit sinoatrial node. Ryanodine (10 μM) reduced the amplitude of action potential-induced intracellular Ca2+([Formula: see text]) transients by 19 ± 3%, increased the time constant for their decay by 51 ± 5%, and slowed spontaneous firing by 32 ± 3%. 1,2-Bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid (BAPTA)-acetoxymethyl ester (AM; 25 μM) inhibited the [Formula: see text] transients and slowed spontaneous firing by 28 ± 4%. Ryanodine did not alter hyperpolarization-activated or time-independent inward current, but it reduced the sum of L- and T-type Ca2+ currents ( I Ca,L and I Ca,T) in both the presence and absence of BAPTA-AM. In contrast, I Ca,L was unchanged by ryanodine. Slow inward current tails, presumed to be Na/Ca exchange current ( I Na/Ca), were abolished by BAPTA or ryanodine. The results suggest that a decrement of I Ca,T, due to reduction of the intracellular Ca2+ concentration or a direct effect of ryanodine on T-type Ca2+channels, contributes to the negative chronotropic effect. Another possibility, based primarily on theory and results in other preparations, is that a reduction of I Na/Ca, as a consequence of the smaller action potential-induced[Formula: see text] transients, contributes to the effect of ryanodine.
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Lee, Rami, Na-Eun Lee, Hongik Hwang, Hyewhon Rhim, Ik-Hyun Cho, and Seung-Yeol Nah. "Ginseng Gintonin Enhances Hyaluronic Acid and Collagen Release from Human Dermal Fibroblasts Through Lysophosphatidic Acid Receptor Interaction." Molecules 24, no. 24 (December 4, 2019): 4438. http://dx.doi.org/10.3390/molecules24244438.
Full textAbstract:
Gintonin is a newly discovered component of ginseng and acts as a ligand for G protein-coupled lysophosphatidic acid (LPA) receptors. It is currently unclear whether gintonin has skin-related effects. Here, we examined the effects of a gintonin-enriched fraction (GEF) on [Ca2+]i transient induction in human dermal fibroblasts (HDFs). We found that GEF treatment transiently induced [Ca2+]i in a dose-dependent manner. GEF also increased cell viability and proliferation, which could be blocked by Ki16425, an LPA1/3 receptor antagonist, or 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), a calcium chelator. We further found that GEF stimulated hyaluronic acid (HA) release from HDFs in a dose- and time-dependent manner, which could be attenuated by Ki16425, U73122, a phospholipase C inhibitor, 2-Aminoethoxydiphenyl borate (2-APB), an IP3 receptor antagonist, and BAPTA-AM. Moreover, we found that GEF increased HA synthase 1 (HAS1) expression in a time-dependent manner. We also found that GEF stimulates collagen release and the expression of collagen 1, 3, and 7 synthases in a time-dependent manner. GEF-mediated collagen synthesis could be blocked by Ki16425, U73122, 2-APB, and BAPTA-AM. GEF treatment also increased the mRNA levels of LPA1-6 receptor subtypes at 8 h and increased the protein levels of LPA1-6 receptor subtypes at 8 h. Overall, these results indicate that the GEF-mediated transient induction of [Ca2+]i is coupled to HA and collagen release from HDFs via LPA receptor regulations. We can, thus, conclude that GEF might exert a beneficial effect on human skin physiology via LPA receptors.
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Brown, D. M., K. Donaldson, P. J. Borm, R. P. Schins, M. Dehnhardt, P. Gilmour, L. A. Jimenez, and V. Stone. "Calcium and ROS-mediated activation of transcription factors and TNF-α cytokine gene expression in macrophages exposed to ultrafine particles." American Journal of Physiology-Lung Cellular and Molecular Physiology 286, no. 2 (February 2004): L344—L353. http://dx.doi.org/10.1152/ajplung.00139.2003.
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Ultrafine (Uf) particles are a component of particulate air pollution suggested to be responsible for the health effects associated with elevations of this pollutant. We have previously suggested that Uf particles, through the induction of oxidative stress, may induce inflammation in the lung, thus exacerbating preexisting illness in susceptible individuals. Alveolar macrophages are considered to play a key role in particlemediated inflammation and lung disease. The effect of Uf particles on rat alveolar macrophages and human blood monocytes was investigated with reference to the roles of calcium and reactive oxygen species (ROS). TNF-α protein release, intracellular calcium concentration, TNF-α mRNA expression, and transcription factor activation were studied as end points after treatment of rat alveolar macrophages or peripheral blood monocytes. The calcium channel blocker verapamil, the intracellular calcium chelator BAPTA-AM, the calmodulin inhibitor W-7, and the antioxidants Trolox and Nacystelin (NAL) were included in combination with Uf particles. Verapamil reduced intracellular calcium concentration in rat alveolar macrophages on stimulation with Uf particles. This effect was also apparent with transcription factor AP-1 activation. All antagonists and antioxidants reduced Uf-stimulated nuclear localization of the p50 and p65 subunits of NF-κB in human monocytes. Verapamil, BAPTA-AM, and NAL reduced Uf-stimulated TNF-α protein release, whereas only verapamil reduced Uf-stimulated mRNA expression in rat alveolar macrophages. In human monocytes, verapamil, Trolox, BAPTA-AM, and W-7 reduced Uf-stimulated TNF-α protein release. These findings suggest that Uf particles may exert proinflammatory effects by modulating intracellular calcium concentrations, activation of transcription factors, and cytokine production through a ROS-mediated mechanism.
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LAWRENCE, Yvonne, Jean Pierre OZIL, and Karl SWANN. "The effects of a Ca2+ chelator and heavy-metal-ion chelators upon Ca2+ oscillations and activation at fertilization in mouse eggs suggest a role for repetitive Ca2+ increases." Biochemical Journal 335, no. 2 (October 15, 1998): 335–42. http://dx.doi.org/10.1042/bj3350335.
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During fertilization in mouse eggs, the sperm triggers a series of intracellular Ca2+ oscillations that lead to egg activation, as indicated by pronuclear formation. We show that Ca2+ oscillations in fertilized mouse eggs can be inhibited by addition of either the Ca2+ chelator 1,2-bis-(o-aminophenoxy)ethane-N,N,N´,N´-tetra-acetic acid acetoxymethyl ester (BAPTA-AM) or the heavy-metal-ion chelator N,N,N´,N´-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) plus dithiothreitol (DTT). Both treatments inhibited Ca2+ oscillations, but they had different effects upon egg activation. Blocking Ca2+ oscillations with BAPTA-AM after the occurrence of just two Ca2+ spikes resulted in most eggs forming pronuclei. However, we found that BAPTA-AM-treated fertilizing eggs showed a decreased rate of protein synthesis, which by itself can promote egg activation. In contrast, blocking Ca2+ oscillations with TPEN plus DTT was accompanied by the inhibition of egg activation with no significant effect on protein synthesis. In eggs that were fertilized and then treated with TPEN plus DTT, there was a correlation between the number of Ca2+ spikes and the proportion of eggs that formed pronuclei, as well as between the number of Ca2+ spikes and the time taken for pronuclear formation and the first mitosis to occur. The addition of TPEN plus DTT did not block the generation of Ca2+ spikes or pronuclear formation when eggs were artificially stimulated by electroporation pulses. These data suggest that TPEN plus DTT inhibits pronuclear formation in fertilizing eggs via the inhibition of Ca2+ oscillations and that the number of Ca2+ spikes may regulate egg activation.
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Reyes, Fredy D., and Edgar T. Walters. "Long-Lasting Synaptic Potentiation Induced by Depolarization Under Conditions That Eliminate Detectable Ca2+ Signals." Journal of Neurophysiology 103, no. 3 (March 2010): 1283–94. http://dx.doi.org/10.1152/jn.00704.2009.
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Activity-dependent alterations of synaptic transmission important for learning and memory are often induced by Ca2+ signals generated by depolarization. While it is widely assumed that Ca2+ is the essential transducer of depolarization into cellular plasticity, little effort has been made to test whether Ca2+-independent responses to depolarization might also induce memory-like alterations. It was recently discovered that peripheral axons of nociceptive sensory neurons in Aplysia display long-lasting hyperexcitability triggered by conditioning depolarization in the absence of Ca2+ entry (using nominally Ca2+-free solutions containing EGTA, “0Ca/EGTA”) or the absence of detectable Ca2+ transients (adding BAPTA-AM, “0Ca/EGTA/BAPTA-AM”). The current study reports that depolarization of central ganglia to ∼0 mV for 2 min in these same solutions induced hyperexcitability lasting >1 h in sensory neuron processes near their synapses onto motor neurons. Furthermore, conditioning depolarization in these solutions produced a 2.5-fold increase in excitatory postsynaptic potential (EPSP) amplitude 1–3 h afterward despite a drop in motor neuron input resistance. Depolarization in 0 Ca/EGTA produced long-term potentiation (LTP) of the EPSP lasting ≥1 days without changing postsynaptic input resistance. When re-exposed to extracellular Ca2+ during synaptic tests, prior exposure to 0Ca/EGTA or to 0Ca/EGTA/BAPTA-AM decreased sensory neuron survival. However, differential effects on neuronal health are unlikely to explain the observed potentiation because conditioning depolarization in these solutions did not alter survival rates. These findings suggest that unrecognized Ca2+-independent signals can transduce depolarization into long-lasting synaptic potentiation, perhaps contributing to persistent synaptic alterations following large, sustained depolarizations that occur during learning, neural injury, or seizures.
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Zaffran, Y., H. Lepidi, P. Bongrand, J. L. Mege, and C. Capo. "F-actin content and spatial distribution in resting and chemoattractant-stimulated human polymorphonuclear leucocytes. Which role for intracellular free calcium?" Journal of Cell Science 105, no. 3 (July 1, 1993): 675–84. http://dx.doi.org/10.1242/jcs.105.3.675.
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Intracellular free calcium concentration ([Ca2+]i) plays a pivotal role for many responses in polymorphonuclear leucocytes (PMNs) stimulated by chemoattractants such as N-formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe). The importance of [Ca2+]i in the morphological polarization was investigated by using calcium-manipulated PMNs. We loaded human PMNs with BAPTA/AM to buffer or chelate [Ca2+]i in the presence or the absence of extracellular calcium by using fluo-3/AM as calcium indicator. The shape changes of PMNs were determined by microscopic examination, and membrane ruffling by right-angle light-scatter changes. Actin polymerization and F-actin distribution were recorded by staining PMNs with bodipy-phallacidin and quantified by quantitative fluorescence microscopy. We found that calcium-free incubation of PMNs loaded or not with 50 microM BAPTA/AM did not modify morphological polarization, membrane ruffling, actin assembly and F-actin distribution of PMNs stimulated with fMet-Leu-Phe, suggesting that these responses were probably functionally linked. It should be noted that incubation of PMNs in calcium-free conditions resulted in a radial distribution of F-actin and a moderate polymerization of actin, but not in morphological polarization of PMNs. Moreover, both calcium-sensitive and calcium-insensitive mechanisms of actin polymerization were additive, and inhibitable by 5 micrograms/ml cytochalasin B.
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Pryor, Paul R., Barbara M. Mullock, Nicholas A. Bright, Sally R. Gray, and J. Paul Luzio. "The Role of Intraorganellar Ca2+In Late Endosome–Lysosome Heterotypic Fusion and in the Reformation of Lysosomes from Hybrid Organelles." Journal of Cell Biology 149, no. 5 (May 29, 2000): 1053–62. http://dx.doi.org/10.1083/jcb.149.5.1053.
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We have investigated the requirement for Ca2+ in the fusion and content mixing of rat hepatocyte late endosomes and lysosomes in a cell-free system. Fusion to form hybrid organelles was inhibited by 1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA), but not by EGTA, and this inhibition was reversed by adding additional Ca2+. Fusion was also inhibited by methyl ester of EGTA (EGTA-AM), a membrane permeable, hydrolyzable ester of EGTA, and pretreatment of organelles with EGTA-AM showed that the chelation of lumenal Ca2+ reduced the amount of fusion. The requirement for Ca2+ for fusion was a later event than the requirement for a rab protein since the system became resistant to inhibition by GDP dissociation inhibitor at earlier times than it became resistant to BAPTA. We have developed a cell-free assay to study the reformation of lysosomes from late endosome–lysosome hybrid organelles that were isolated from the rat liver. The recovery of electron dense lysosomes was shown to require ATP and was inhibited by bafilomycin and EGTA-AM. The data support a model in which endocytosed Ca2+ plays a role in the fusion of late endosomes and lysosomes, the reformation of lysosomes, and the dynamic equilibrium of organelles in the late endocytic pathway.
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Xie, Ying, Yue Han, De Pei Wu, Aining Sun, and Wei Zhang. "Influence of Protein Kinase C and Calcium in the Course of Thrombin Receptors Activation." Blood 110, no. 11 (November 16, 2007): 3194. http://dx.doi.org/10.1182/blood.v110.11.3194.3194.
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Abstract Object In order to compare the functions of protein kinase C (PKC) and calcium (Ca2+) in platelet aggregation and platelet membrane surface glycoproteins GPIb expression after thrombin receptors activation, then to investigate the role of Gq signal transmission pathway in the course of thrombin receptors activation. Methods Peptide SFLLRN (PAR1-AP) and AYPGKF (PAR4-AP) were used for stimulating platelet at different time point (0, 1, 2, 5, 10, 30min), then the alterations of platelet aggregation and GPIb were analyzed in the involvement of Ro-31-2220 (inhibitor of PKC) and BAPTA/AM (calcium chelator). Results Either PAR1 or PAR4 peptide can induce absolute platelet aggregation, together with a reversible internalization of GPIb. Platelet aggregation was inhibited by Ro-31-2220 or BAPTA/AM while the shape change curve still occurred upon PARs activation. In addition, Ro-31-2220 decreases GPIb centralisation upon PAR1 stimulation (P <0.05 at 1, 2 min), though it blocks the pool of GPIb inside platelet in PAR4 activation (P <0.05 at 10, 30 min). Meanwhile, GPIb internalization was blocked by BAPTA for both peptides (P <0.05 at 1∼10 min). Conclusion All the results confirm a critical role of Gq pathway in thrombin signal transmission through the involvement of protein kinase C and calcium. Calcium is closely correlated with the thrombin receptors activation, seemed to be similar for two PARs signal pathways. Protein kinase C urges GPIb centralisation in PAR1 pathway and accelerates GPIbα return in PAR4 pathway.
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Sandvik, A. K., E. Brenna, and H. L. Waldum. "Calcium mediates gastrin-induced gastric histamine release in the rat." American Journal of Physiology-Gastrointestinal and Liver Physiology 264, no. 1 (January 1, 1993): G51—G56. http://dx.doi.org/10.1152/ajpgi.1993.264.1.g51.
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This study examined the second messenger system responsible for gastrin-induced histamine release from the rat stomach. We examined the effect of different concentrations of ionized calcium, the calcium-channel blockers verapamil and nicardipine, and the intracellular calcium-chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl ester (BAPTA/AM) on gastrin-stimulated histamine release in the totally isolated vascularly perfused rat stomach. Moreover, the effect on baseline histamine release of caffeine as well as of forskolin and 3-isobutyl-1-methylxanthine (IBMX) was tested. Gastrin induced an immediate 10- to 15-fold increase in venous histamine. Perfusate ionized calcium in the 0.25-1.25 mM range did not affect histamine release; histamine release was attenuated by the 0.00 and 1.75 mM calcium concentrations. Verapamil, nicardipine, and BAPTA/AM inhibited gastrin-stimulated histamine release. Caffeine stimulated the release, whereas forskolin and IBMX had no effect. We conclude that gastrin-induced histamine release from the rat stomach is mediated by calcium, probably both from the intracellular pool and by transmembrane flux from the extracellular space.
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Glaser, Shannon, Domenico Alvaro, Tania Roskams, Jo Lynne Phinizy, George Stoica, Heather Francis, Yoshiyuki Ueno, et al. "Dopaminergic inhibition of secretin-stimulated choleresis by increased PKC-γ expression and decrease of PKA activity." American Journal of Physiology-Gastrointestinal and Liver Physiology 284, no. 4 (April 1, 2003): G683—G694. http://dx.doi.org/10.1152/ajpgi.00302.2002.
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To determine the role and mechanisms of action by which dopaminergic innervation modulates ductal secretion in bile duct-ligated rats, we determined the expression of D1, D2, and D3 dopaminergic receptors in cholangiocytes. We evaluated whether D1, D2 (quinelorane), or D3 dopaminergic receptor agonists influence basal and secretin-stimulated choleresis and lumen expansion in intrahepatic bile duct units (IBDU) and cAMP levels in cholangiocytes in the absence or presence of BAPTA-AM, chelerythrine, 1-(5-isoquinolinylsulfonyl)-2-methyl piperazine (H7), or rottlerin. We evaluated whether 1) quinelorane effects on ductal secretion were associated with increased expression of Ca2+-dependent PKC isoforms and 2) increased expression of PKC causes inhibition of PKA activity. Quinelorane inhibited secretin-stimulated choleresis in vivo and IBDU lumen space, cAMP levels, and PKA activity in cholangiocytes. The inhibitory effects of quinelorane on secretin-stimulated ductal secretion and PKA activity were blocked by BAPTA-AM, chelerythrine, and H7. Quinelorane effects on ductal secretion were associated with activation of the Ca2+-dependent PKC-γ but not other PKC isoforms. The dopaminergic nervous system counterregulates secretin-stimulated ductal secretion in experimental cholestasis.
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Baliño, Pablo, Lidón Monferrer, Raúl Pastor, and Carlos M. G. Aragon. "Intracellular calcium chelation with BAPTA-AM modulates ethanol-induced behavioral effects in mice." Experimental Neurology 234, no. 2 (April 2012): 446–53. http://dx.doi.org/10.1016/j.expneurol.2012.01.016.
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Boeynaems, Jean-Marie, Sylvie Heilporn, Fabienne Broeders, and Jean-Claude Braekman. "Enhancement of the endothelial production of prostacyclin by substituted derivatives of BAPTA-AM." European Journal of Pharmacology 233, no. 1 (March 1993): 13–20. http://dx.doi.org/10.1016/0014-2999(93)90343-g.
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Wu, Shuaishuai, Igor F. Canisso, Weigang Yang, Ihteshamu Ul Haq, Qiang Liu, Ying Han, and Shenming Zeng. "Intracellular calcium chelating agent (BAPTA-AM) aids stallion semen cooling and freezing-thawing." Reproduction in Domestic Animals 53, no. 5 (July 9, 2018): 1235–42. http://dx.doi.org/10.1111/rda.13245.
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Heilporn, S., F. Broeders, D. Daloze, J. C. Braekman, and J. M. Boeynaems. "Synthesis of BAPTA-AM Analogues Capable of Enhancing the Vascular production of Prostacyclin." Bulletin des Sociétés Chimiques Belges 103, no. 7-8 (September 1, 2010): 309–19. http://dx.doi.org/10.1002/bscb.19941030703.
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STRAYER, David S., Jan B. HOEK, Andrew P. THOMAS, and Martyn K. WHITE. "Cellular activation by Ca2+ release from stores in the endoplasmic reticulum but not by increased free Ca2+ in the cytosol." Biochemical Journal 344, no. 1 (November 8, 1999): 39–46. http://dx.doi.org/10.1042/bj3440039.
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Ca2+ release from intracellular stores and/or transmembrane influx can increase the cytosolic free Ca2+ concentration ([Ca2+]i). Such changes in [Ca2+]i might transduce signals regulating transcription, motility, secretion, and so on. Surfactant secretagogues such as ATP and ionomycin stimulate the release and transmembrane influx of Ca2+, both of which increase [Ca2+]i. The addition of surfactant protein A (SP-A) or depleting cellular Ca2+ inhibited both surfactant secretion and Ca2+ transients. Current results suggest that Ca2+ signalling stimulates surfactant secretion by type II pneumocytes, but not via increased [Ca2+]i. Treatment of cells with a Ca2+ chelator, bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid acetoxymethyl ester (BAPTA-AM), stimulated secretion but decreased [Ca2+]i. Adding SP-A or depleting Ca2+ inhibited BAPTA-AM-induced secretion. When studied directly, Ca2+ in the endoplasmic reticulum store ([Ca2+]l) decreased in response to BAPTA, ionomycin and thapsigargin, and increased in response to SP-A. Phorbol ester (PMA) induced surfactant secretion without altering [Ca2+]i or [Ca2+]l and was unaffected by Ca2+ depletion. The addition of PMA to Ca2+-releasing secretagogues increased secretion, but combining two Ca2+-releasing secretagogues did not. These results suggest that (1) Ca2+ signalling of type II cell surfactant secretion reflects changes in [Ca2+]l, not [Ca2+]i, (2) PMA elicits secretion differently from Ca2+-releasing secretagogues, and (3) SP-A inhibits secretion by enhancing Ca2+ sequestration within endoplasmic reticulum stores. Whether other cell types signal via changes in [Ca2+]l is unknown.
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Oliveira-Souza, M., and M. De Mello-Aires. "Interaction of angiotensin II and atrial natriuretic peptide on pHi regulation in MDCK cells." American Journal of Physiology-Renal Physiology 279, no. 5 (November 1, 2000): F944—F953. http://dx.doi.org/10.1152/ajprenal.2000.279.5.f944.
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The effect of ANG II and atrial natriuretic peptide (ANP) on intracellular pH (pHi) and cytosolic free calcium concentration ([Ca2+]i) was investigated in Madin-Darby canine kidney cells by using the fluorescent probes 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl ester (AM) and fura 2-AM or fluo 4-AM. pHi recovery rate was examined in the first 2 min after the acidification of pHi with a NH4Cl pulse. In the control situation, the pHi recovery rate was 0.088 ± 0.014 pH units/min ( n = 14); in the absence of external Na+, this value was decreased. ANG II (10−12or 10−9 M) caused an increase in this value, but ANG II (10−7 M) decreased it. ANP (10−6 M) or dimethyl-1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid (BAPTA)-AM (50 μM) alone did not affect this value but impaired both stimulatory and inhibitory effects of ANG II. ANG II (10−12, 10−9, or 10−7 M) increased [Ca2+]i progressively from 99 ± 10 ( n = 20) to 234 ± 7 mM ( n = 10). ANP or dimethyl-BAPTA-AM decreases [Ca2+]i, and the subsequent addition of ANG II caused a recovery of [Ca2+]i but without reaching ANG II values found in the absence of these agents. The results indicate a role for [Ca2+]i in regulating the process of pHi recovery mediated by the Na+/H+ exchanger, stimulated/impaired by ANG II, and not affected by ANP or ANG II plus ANP. This hormonal interaction may represent physiologically relevant regulation in conditions of volume alterations in the intact animal.
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Siflinger-Birnboim, A., H. Lum, P. J. Del Vecchio, and A. B. Malik. "Involvement of Ca2+ in the H2O2-induced increase in endothelial permeability." American Journal of Physiology-Lung Cellular and Molecular Physiology 270, no. 6 (June 1, 1996): L973—L978. http://dx.doi.org/10.1152/ajplung.1996.270.6.l973.
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We studied the role of Ca2+ in mediating the hydrogen peroxide (H2O2)-induced increase in endothelial permeability to 125I-labeled albumin using bovine pulmonary microvessel endothelial cells (BMVEC). Changes in cytosolic-free Ca2+ ([Ca2+]i) were monitored in BMVEC monolayers loaded with the Ca(2+)-sensitive membrane permeant fluorescent dye fura 2-AM. H2O2 (100 microM) produced a rise in [Ca2+]i within 10 s that was reduced by the addition of EGTA to the medium. Uptake of 45Ca2+ from the extracellular medium increased in the presence of H2O2 (100 microM) compared with control monolayers, suggesting that the H2O2-induced rise in [Ca2+]i is partly the result of extracellular Ca2+ influx. The effects of [Ca2+]i on endothelial permeability were addressed by pretreatment of BMVEC monolayers with BAPTA-AM (3-5 microM), a membrane permeant Ca2+ chelator, before the H2O2 exposure. BAPTA-AM produced an approximately 50% decrease in the H2O2-induced increase in endothelial permeability compared with endothelial cell monolayers exposed to H2O2 alone. The increase in endothelial permeability was independent of Ca2+ influx, since LaCl3 (0-100 microM), which displaces Ca2+ from binding sites on the cell surface, did not modify the permeability response. These results indicate that the rise in [Ca2+]i produced by H2O2 is a critical determinant of the increase in endothelial permeability.
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Yang, Won Seok, Soo Young Moon, Mee Jeong Lee, and Su-Kil Park. "Epigallocatechin-3-Gallate Attenuates the Effects of TNF-α in Vascular Endothelial Cells by Causing Ectodomain Shedding of TNF Receptor 1." Cellular Physiology and Biochemistry 38, no. 5 (2016): 1963–74. http://dx.doi.org/10.1159/000445557.
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Background/Aims: We investigated the mechanism underlying anti-tumor necrosis factor-α (TNF-α) effects of epigallocatechin-3-gallate (EGCG) in human aortic endothelial cells. Methods: Tumor necrosis factor receptor 1 (TNFR1) was assessed by Western blot analysis. Cytosolic Ca2+ was measured using Fluo-4 AM. A disintegrin and metalloprotease 10 (ADAM10) was localized by immunofluorescence staining. Results: EGCG caused ectodomain shedding of TNFR1 within 30 min and attenuated TNF-α-induced endothelin-1 (ET-1) expression. EGCG-induced TNFR1 ectodomain shedding was prevented by BAPTA-AM (intracellular Ca2+ chelator), but not by the absence of extracellular Ca2+. In physiologic extracellular Ca2+ concentration, EGCG markedly increased cytosolic Ca2+. Even in the absence of extracellular Ca2+, EGCG raised cytosolic Ca2+, though less potently. siRNA depletion of ADAM10 prevented EGCG-induced ectodomain shedding of TNFR1 and also diminished the inhibitory effect of EGCG on TNF-α-induced ET-1 expression. EGCG caused translocation of ADAM10 to the plasma membrane, and this effect was prevented by BAPTA-AM. Besides extracellular Ca2+ influx, release of intracellular stored Ca2+ caused ADAM10-dependent ectodomain shedding of TNFR1. Conclusion: EGCG decreases the responsiveness of cells to TNF-α by causing ADAM10-dependent ectodomain shedding of TNFR1. This effect was attributed to its property to increase cytosolic Ca2+ through both extracellular Ca2+ influx and release of stored Ca2+.
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Berchtold, Craig M., Zhao-Hui Wu, Tony T. Huang, and Shigeki Miyamoto. "Calcium-Dependent Regulation of NEMO Nuclear Export in Response to Genotoxic Stimuli." Molecular and Cellular Biology 27, no. 2 (October 30, 2006): 497–509. http://dx.doi.org/10.1128/mcb.01772-06.
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ABSTRACT The mechanisms involved in activation of the transcription factor NF-κB by genotoxic agents are not well understood. Previously, we provided evidence that a regulatory subunit of the IκB kinase (IKK) complex, NF-κB essential modulator (NEMO)/IKKγ, is a component of a nuclear signal that is generated after DNA damage to mediate NF-κB activation. Here, we found that etoposide (VP16) and camptothecin induced increases in intracellular free calcium levels at 60 min after stimulation of CEM T leukemic cells. Inhibition of calcium increases by calcium chelators, BAPTA-AM and EGTA-AM, abrogated NF-κB activation by these agents in several cell types examined. Conversely, thapsigargin and ionomycin attenuated the BAPTA-AM effects and promoted NF-κB activation by the genotoxic stimuli. Analyses of nuclear NEMO levels in VP16-treated cells suggested that calcium was required for nuclear export of NEMO. Inhibition of the nuclear exporter CRM1 by leptomycin B did not interfere with NEMO nuclear export. Similarly, deficiency of a plausible calcium-dependent nuclear export receptor, calreticulin, failed to prevent NF-κB activation by VP16. However, temperature inactivation of the Ran guanine nucleotide exchange factor RCC1 in the tsBN2 cell line harboring a temperature-sensitive mutant of RCC1 blocked NF-κB activation induced by genotoxic stimuli. Overexpression of Ran in this cell model showed that DNA damage stimuli induced formation of a complex between Ran and NEMO, suggesting that RCC1 regulated NF-κB activation through the modulation of RanGTP. Indeed, evidence for VP16-inducible interaction between Ran-GTP and NEMO could be obtained by means of glutathione S-transferase (GST) pull-down assays using GST fused to the Ran binding domain of RanBP2, which specifically interacts with the GTP-bound form of Ran. BAPTA-AM did not alter these interactions, suggesting that calcium is a necessary step beyond the formation of a Ran-GTP-NEMO complex in the nucleus. These results suggest that calcium has a unique role in genotoxic stress-induced NF-κB signaling by regulating nuclear export of NEMO subsequent to the formation of a nuclear export complex composed of Ran-GTP, NEMO, and presumably, an undefined nuclear export receptor.
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Burlando, Bruno, Barbara Marchi, Isabella Panfoli, and Aldo Viarengo. "Essential role of Ca2+-dependent phospholipase A2in estradiol-induced lysosome activation." American Journal of Physiology-Cell Physiology 283, no. 5 (November 1, 2002): C1461—C1468. http://dx.doi.org/10.1152/ajpcell.00429.2001.
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The mechanism of lysosome activation by 17β-estradiol has been studied in mussel blood cells. Cell treatment with estradiol induced a sustained increase of cytosolic free Ca2+that was completely prevented by preincubating the cells with the Ca2+chelator BAPTA-AM. Estradiol treatment was also followed by destabilization of the lysosomal membranes, as detected in terms of the lysosomes' increased permeability to neutral red. The effect of estradiol on lysosomes was almost completely prevented by preincubation with the inhibitor of cytosolic Ca2+-dependent PLA2(cPLA2), arachidonyl trifluoromethyl ketone (AACOCF3), and was significantly reduced by preincubation with BAPTA-AM. In contrast, it was virtually unaffected by preincubation with the inhibitor of Ca2+-independent PLA2, ( E)-6-(bromomethylene)tetrahydro-3-(1-naphtalenyl)-2 H-pyran-2-one (BEL). The Ca2+ionophore A-23187 yielded similar effects on [Ca2+]iand lysosomes. Exposure to estradiol also resulted in cPLA2translocation from cytosol to membranes, lysosome enlargement, and increased protein degradation. These results suggest that the destabilization of lysosomal membranes following cell exposure to estradiol occurs mainly through a Ca2+-dependent mechanism involving activation of Ca2+-dependent PLA2. This mechanism promotes lysosome fusion and catabolic activities and may mediate short-term estradiol effects.
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Alpini, Gianfranco, Noriatsu Kanno, Jo Lynne Phinizy, Shannon Glaser, Heather Francis, Silvia Taffetani, and Gene LeSage. "Tauroursodeoxycholate inhibits human cholangiocarcinoma growth via Ca2+-, PKC-, and MAPK-dependent pathways." American Journal of Physiology-Gastrointestinal and Liver Physiology 286, no. 6 (June 2004): G973—G982. http://dx.doi.org/10.1152/ajpgi.00270.2003.
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Tauroursodeoxychate (TUDCA) is used for the treatment of cholangiopathies including primary sclerosing cholangitis, which is considered the primary risk factor for cholangiocarcinoma. The effect of TUDCA on cholangiocarcinoma growth is unknown. We evaluated the role of TUDCA in the regulation of growth of the cholangiocarcinoma cell line Mz-ChA-1. TUDCA inhibited the growth of Mz-ChA-1 cells in concentration- and time-dependent manners. TUDCA inhibition of cholangiocarcinoma growth was blocked by BAPTA-AM, an intracellular Ca2+ concentration ([Ca2+]i) chelator, and H7, a PKC-α inhibitor. TUDCA increased [Ca2+]i and membrane translocation of the Ca2+-dependent PKC-α in Mz-ChA-1 cells. TUDCA inhibited the activity of MAPK, and this inhibitory effect of TUDCA was abrogated by BAPTA-AM and H7. TUDCA did not alter the activity of Raf-1 and B-Raf and the phosphorylation of MAPK p38 and JNK/stress-activated protein kinase. TUDCA inhibits Mz-ChA-1 growth through a signal-transduction pathway involving MAPK p42/44 and PKC-α but independent from Raf proteins and MAPK p38 and JNK/stress-activated protein kinases. TUDCA may be important for the treatment of cholangiocarcinoma.
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Stachecki, J. J., and D. R. Armant. "Transient release of calcium from inositol 1,4,5-trisphosphate-specific stores regulates mouse preimplantation development." Development 122, no. 8 (August 1, 1996): 2485–96. http://dx.doi.org/10.1242/dev.122.8.2485.
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Inositol 1,4,5-trisphosphate can regulate growth and differentiation by modulating the release of intracellular Ca2+ in a variety of cellular systems, and it is involved in oocyte activation. Recent studies suggest that mammalian preimplantation development may also be regulated by the release of Ca2+ from intracellular stores. The rate of cavitation and cell division was accelerated after a transient elevation of intracellular Ca2+ levels was induced in morulae by exposure to ethanol or ionomycin. Embryos exposed to BAPTA-AM, a chelator of intracellular Ca2+, exhibited a brief dose-dependent reduction in basal Ca2+ levels, a temporal inhibition of ionophore-induced Ca2+ signalling and a subsequent delay in blastocoel formation. BAPTA-AM at 0.5 microM did not significantly alter the basal intracellular calcium level, but chelated Ca2+ that was released after ethanol exposure and thereby attenuated the ethanol-induced acceleration of cavitation. BAPTA-AM also inhibited cell division to the 16-cell stage in a dose-dependent manner, which correlated with the inhibition of cavitation. Thimerosal and inositol 1,4,5-trisphosphate significantly elevated the intracellular Ca2+ concentration in mouse morula-stage embryos, providing evidence for the existence of inositol 1,4,5-trisphosphate-sensitive Ca2+ stores. Although caffeine failed to release intracellular Ca2+, ryanodine induced a small biphasic release of Ca2+, suggesting that ryanodine-sensitive Ca2+ stores may also exist in mouse embryos. Morulae exposed to the calmodulin inhibitor W-7 exhibited a dose-dependent delay in blastocoel formation. A 4 hour exposure to 10 microM W-7 did not significantly alter cavitation, but attenuated the ionophore-induced stimulation of blastocoel formation. This finding suggests that the developmental effects produced through Ca2+ signalling are mediated by calmodulin. Our results demonstrate that Ca2+ release in mouse morulae occurs predominantly through the inositol 1,4,5-trisphosphate receptor, and that alteration of intracellular Ca2+ levels can accelerate or delay embryonic growth and differentiation, providing a mechanistic link between the regulation of oocyte and embryonic development.