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1
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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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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WU, Qiang-Sheng, Qiu-Dan NI, Yan-Cong QUE, and Wei HUANG. "Calcium and Calmodulin Involve in Mycorrhizal and Root Development in Trifoliate Orange Colonized by Rhizophagus intraradices." Notulae Botanicae Horti Agrobotanici Cluj-Napoca 42, no. 2 (December 2, 2014): 380–85. http://dx.doi.org/10.15835/nbha4229635.
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A pot experiment was made to study effects of ethylene glycol tetraacetic acid (EGTA, an inhibitor of Ca2+) and trifluoperazine (TFP, an inhibitor of calmodulin (CaM) on mycorrhizal colonization, growth performance, and chlorophyll, sucrose and glucose concentrations of four-month-old trifoliate orange (Poncirus trifoliata) seedlings under mycorrhization with Rhizophagus intraradices. Exogenous EGTA and TFP notably inhibited root mycorrhizal colonization, and the addition of EGTA also decreased soil hyphal length. In general, EGTA treatment decreased but TFP increased easily extractable glomalin-related soil protein (EE-GRSP) and total GRSP (T-GRSP) concentrations. In addition, EGTA and TFP applications generally significantly inhibited growth performance (height, stem diameter, leaf number, and shoot and root biomass), root traits (total length, surface area, volume, and number of 1st, 2nd and 3rd order lateral root), and chlorophyll a,b and a+b concentrations, the mycorrhizal inoculation generally reversed the negative effects and markedly increased these variables, irrespective of whether the seedlings were applied by inhibitors or not. EGTA and TFP treatments generally inhibited sucrose and glucose levels of leaf and root, except that TFP application notably increased root glucose in AM and non-AM seedlings. AMF inoculation resulted in carbohydrate modification: decrease in leaf sucrose, increase in root sucrose and leaf glucose, as well increase in root glucose under no-inhibitor and EGTA conditions and decrease in root glucose under TFP. It suggests that Ca2+ and CaM were involved in mycorrhizal and root development in trifoliate orange.
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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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Johnson, J. D., Y. Jiang, and M. Flynn. "Modulation of Ca2+ transients and tension by intracellular EGTA in intact frog muscle fibers." American Journal of Physiology-Cell Physiology 272, no. 5 (May 1, 1997): C1437—C1444. http://dx.doi.org/10.1152/ajpcell.1997.272.5.c1437.
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Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) was used as an intracellular Ca2+ chelator to shorten the duration of the Ca2+ transient and to determine the rate-limiting steps in relaxation of frog skeletal muscle at 10 and 20 degrees C. Incubation with the acetoxymethyl ester of EGTA (EGTA-AM) produced a linear approximately twofold increase in the rate of fall in twitch Ca2+ concentration ([Ca2+]) over 0-70 min at 10 degrees C. The rate of relaxation initially increased from 5 to 9/s over 0-13 min and then leveled, as the rate of fall in [Ca2+] continued to increase and peak force decreased. Increasing the rate of fall in [Ca2+] increased the rate of relaxation, until a rate-limiting step was reached at approximately 9/s at 10 degrees C. At 20 degrees C, incubation with EGTA-AM produced a linear approximately twofold increase in the rate of fall in twitch [Ca2+] without affecting the rate of relaxation (27/s). Ca2+ dissociated from the regulatory sites of purified troponin (Tn) at approximately 8/s at 10 degrees C and at 23/s at 20 degrees C. When the duration of the Ca2+ transient was decreased by EGTA or increased by partial inhibition of the sarcoplasmic reticulum Ca(2+)-ATPase, twitch force increased linearly with Ca2+ transient duration. Thus the duration of the Ca2+ transient is a primary determinant of twitch force and, whereas the rate of fall in [Ca2+] is rate limiting for relaxation at 10 degrees C, Ca2+ dissociation from Tn may be rate limiting at 20 degrees C.
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Uemura, Naomi, Kenneth E. J. Dickinson, Yuji Horiguchi, Haruki Matsumoto, and Basil I. Hirschowitz. "Decreased secretion due to a Ca2+ influx defect in frog peptic cells isolated with EGTA." American Journal of Physiology-Gastrointestinal and Liver Physiology 258, no. 4 (April 1, 1990): G542—G551. http://dx.doi.org/10.1152/ajpgi.1990.258.4.g542.
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Cells prepared from frog esophageal peptic glands by dispersal in low-Ca2+ medium (peptic acini) or 1 mM ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)-containing medium ("EGTA cells") were compared. EGTA cells were characterized by decreased secretory responses to agonists [bombesin (BB), acetylcholine, and isoproterenol] and intracellular messenger activators (forskolin, 12-O-tetradecanoyl-phorbol-13-acetate), decreased relative intrinsic efficacies of muscarinic agonists, and somatostatin insensitivity. Decreased BB and muscarinic receptor responses were not associated with changes in receptor number or characteristics. The time course of BB- and acetylcholine-stimulated pepsinogen secretion indicated that the marked reduction was confined largely to the late secretory phase (2–30 min), dependent on extracellular Ca2+, rather than early phase (2 min) secretion, which is related to release of intracellular Ca2+. The defect could be reversed by the Ca2+ ionophore A23187. BB-stimulated intracellular Ca2+ mobilization measured with fura-2/AM was similar in the two cell preparations, whereas BB-stimulated 45Ca2+ uptake was reduced threefold in EGTA cells, and this defect was also reversed by A23187. Somatostatin inhibited both BB-stimulated secretion and 45Ca uptake by peptic acini, but it had no significant effect on these parameters in EGTA cells. Cytochalasin B inhibited BB stimulation in peptic acini but not EGTA cells. These findings suggest that peptic cells isolated with EGTA exhibit decreased secretory responses that are due at least in part to impairment of a mechanism for uptake of extracellular Ca2+.
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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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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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Ichimura, Hideo, Kaushik Parthasarathi, Jens Lindert, and Jahar Bhattacharya. "Lung surfactant secretion by interalveolar Ca2+ signaling." American Journal of Physiology-Lung Cellular and Molecular Physiology 291, no. 4 (October 2006): L596—L601. http://dx.doi.org/10.1152/ajplung.00036.2006.
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Although clusters of alveoli form the acinus, which is the most distal respiratory unit, it is not known whether interalveolar communication coordinates acinar surfactant secretion. To address this, we applied real-time digital imaging in conjunction with photo-excited Ca2+ uncaging in intact alveoli of the isolated, blood-perfused rat lung. We loaded alveolar cells with the Ca2+ cage o-nitrophenyl EGTA-AM (NP-EGTA-AM) together with the fluorophores, fluo 4, or LysoTracker green (LTG) to determine, respectively, the cytosolic Ca2+ concentration ([Ca2+]cyt) or type 2 cell secretion. To uncage Ca2+ from NP-EGTA, we exposed a region in a selected alveolus to high-intensity UV illumination. As a result, fluo 4 fluorescence increased, whereas LTG fluorescence decreased, in the photo-targeted region, indicating that uncaging both increased [Ca2+]cyt and induced secretion. Concomitantly, [Ca2+]cyt increases conducted from the uncaging site induced type 2 cell secretion in both the selected alveolus as well as in neighboring alveoli, indicating the presence of interalveolar communication. These conducted responses were inhibited by pretreating alveoli with the connexin43 (Cx43)-inhibiting peptides gap 26 and gap 27. However, although the conducted [Ca2+]cyt increase diminished with distance from the uncaging site, type 2 cell secretion rates were similar at all locations. We conclude that Cx43-dependent, interalveolar Ca2+ signals regulate type 2 cell secretion in adjacent alveoli. Such interalveolar communication might facilitate acinar coordination of alveolar function.
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Feller, M. B., K. R. Delaney, and D. W. Tank. "Presynaptic calcium dynamics at the frog retinotectal synapse." Journal of Neurophysiology 76, no. 1 (July 1, 1996): 381–400. http://dx.doi.org/10.1152/jn.1996.76.1.381.
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1. We characterized the kinetics of presynaptic Ca2+ ion concentration in optic nerve fibers and terminals of the optic tectum in Rana pipiens with the use of microfluorimetry. Isolated frog brains were incubated with the membrane-permeant tetraacetoxymethyl ester (AM) of the Ca2+ indicator fura-2. An optic nerve shock caused a transient decrease in the 380-nm excited fluorescence in the optic tectum with a rise time of <15 ms and a recovery to prestimulus levels on a time scale of seconds. 2. In normal saline, the amplitude of the fluorescence transients was dependent on stimulus intensity and at all levels it was directly correlated with the amplitude of postsynaptic field potentials produced by activation of unmyelinated optic nerve fibers. In the presence of the non-N-methyl-D-aspartate glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione, the amplitude and time course of fluorescence transients remained essentially unchanged while postsynaptic field potential amplitude was greatly reduced. Replacing extracellular Ca2+ with Ba2+ blocked unfacilitated postsynaptic field potentials while fluorescence transients remained significant. In reduced-Ca2+ salines (<1 mM), the amplitude of fluorescence transients increased approximately linearly with extracellular [Ca2+], whereas the amplitude the corresponding field potential was nonlinearly related to the fluorescent transient amplitude (approximately 2.5 power). In thin sections of labeled tecta, fluorescence labeling was localized to 1-micron puncta in the termination zone of optic nerve fibers in the superficial layers. Taken together, these results provide strong evidence that the fluorescence transients correspond to an increase in Ca2+ in presynaptic terminals of unmyelinated optic nerve fibers. 3. During trains of optic nerve stimulation, the amplitude of fluorescence transients to succeeding action potentials became smaller. The decrement of the amplitudes was not observed in mag-fura-5-labeled tecta, when the intracellular Ca2+ buffering capacity of fura-2-labeled terminals was increased by incubation with bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA)-AM or ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)-AM, or in low-Ca2+ saline. We conclude that the Ca2+ influx per action potential is constant during the train and that the reduced response was produced by saturation of the fura-2. We provide a mathematical analysis of this saturation effect and use it to estimate the Ca2+ change per action potential. 4. Both BAPTA-AM and EGTA-AM reduced the overall amplitude of fura-2-measured Ca2+ transients and reduced the saturation effect in action potential trains. However, there was a qualitative difference in their effects on the shape of the transient. Incubation with the fast buffer BAPTA prolonged the decay to baseline. In contrast, the slow buffer EGTA (or EDTA) produced an initial decay faster than the control condition while also producing the slower subsequent phase observed with BAPTA. We demonstrate that these results are consistent with numerical simulations of Ca2+ dynamics in a single-compartment model where the fast initial decay is produced by the forward rate of Ca2+ binding to EGTA. 5. Ca2+ influx into tectal presynaptic structures, and also into unmyelinated axons in the isolated optic nerve, was diminished (60-70%) in the presence of the voltage-activated Ca2+ channel blocker omega-conotoxin GVIA, but was only weakly affected (approximately 10%) by omega-agatoxin IVA. 6. After 10- to 50-Hz stimulus trains, synaptic enhancement of unmyelinated fibers decayed with a characteristic time similar to fura-2 fluorescence decays. Incubation with EDTA-AM or EGTA-AM produced little effect on evoked release but reduced both the amplitude of the fura-2-measured Ca2+ transient and the amplitude of short-term synaptic enhancement.
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Iseki, R., Y. Kudo, and M. Iwata. "Early mobilization of Ca2+ is not required for glucocorticoid-induced apoptosis in thymocytes." Journal of Immunology 151, no. 10 (November 15, 1993): 5198–207. http://dx.doi.org/10.4049/jimmunol.151.10.5198.
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Abstract Contradictory results have been reported on the question of the role of Ca2+ in glucocorticoid-induced apoptosis in thymocytes. To resolve this problem, we investigated the effect of dexamethasone, a synthetic glucocorticoid, on intracellular Ca2+ concentration ([Ca2+]i), by microscopic fluorometry that enables us to monitor real-time [Ca2+]i of cells loaded with fura-2, a fluorescent Ca2+ indicator, on a single cell basis. The results indicated that dexamethasone does not induce an increase in [Ca2+]i above control level both in murine and rat thymocytes at least for 1 h after the start of the culture. We also investigated whether the depletion of extracellular Ca2+ with EGTA or buffering intracellular Ca2+ with quin-2/AM inhibited glucocorticoid-induced apoptosis as reported on rat thymocytes. Dexamethasone-induced apoptosis in both murine and rat thymocytes, however, was not inhibited by EGTA. High concentrations (25 microM and over) of quin-2/AM inhibited DNA fragmentation, but failed to inhibit cytolysis. Calmodulin inhibitors, trifluoperazine and calmidazolium, also inhibited DNA fragmentation as reported, although they markedly enhanced cytolysis. Therefore, glucocorticoid-induced death is not inhibited by quin-2/AM or calmodulin inhibitors. Furthermore, we have previously found that a proper combination of the calcium ionophore, ionomycin, and the protein kinase activator, PMA, inhibits corticosterone-induced apoptosis. These results suggest that an early increase in [Ca2+]i is neither induced by glucocorticoids nor responsible for glucocorticoid-induced apoptosis in thymocytes.
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Abdrakhmanov, M. M., A. M. Petrov, P. N. Grigoryev, and A. L. Zefirov. "Depolarization-Induced Calcium-Independent Synaptic Vesicle Exo- and Endocytosis at Frog Motor Nerve Terminals." Acta Naturae 5, no. 4 (December 15, 2013): 77–82. http://dx.doi.org/10.32607/20758251-2013-5-4-77-82.
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The transmitter release and synaptic vesicle exo- and endocytosis induced by constant current depolarization of nerve terminals were studied by microelectode extracellular recording of miniature endplate currents and fluorescent microscopy (FM 1-43 styryl dye). Depolarization of the plasma membrane of nerve terminals in the control specimen was shown to significantly increase the MEPC frequency (quantal transmitter release) and exocytotic rate (FM 1-43 unloading from the synaptic vesicles preliminarily stained with the dye), which was caused by a rise in the intracellular Ca 2+ concentration due to opening of voltage-gated Ca channels. A slight increase in the MEPC frequency and in the rate of synaptic vesicle exocytosis was observed under depolarization in case of blockade of Ca channels and chelating of intracellular Ca 2+ ions (cooperative action of Cd 2+ and EGTA-AM). The processes of synaptic vesicle endocytosis (FM 1-43 loading) were proportional to the number of synaptic vesicles that had undergone exocytosis both in the control and in case of cooperative action of Cd 2+ and EGTA-AM. A hypothesis has been put forward that Ca-independent synaptic vesicle exo- and endocytosis that can be induced directly by depolarization of the membrane exists in the frog motor terminal in addition to the conventional Ca-dependent process.
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Losavio, Adriana, and S. Muchnik. "Spontaneous acetylcholine release in mammalian neuromuscular junctions." American Journal of Physiology-Cell Physiology 273, no. 6 (December 1, 1997): C1835—C1841. http://dx.doi.org/10.1152/ajpcell.1997.273.6.c1835.
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Spontaneous secretion of the neurotransmitter acetylcholine in mammalian neuromuscular synapsis depends on the Ca2+ content of nerve terminals. The Ca2+ electrochemical gradient favors the entry of this cation. We investigated the possible involvement of three voltage-dependent Ca2+ channels (VDCC) (L-, N-, and P/Q-types) on spontaneous transmitter release at the rat neuromuscular junction. Miniature end-plate potential (MEPP) frequency was clearly reduced by 5 μM nifedipine, a blocker of the L-type VDCC, and to a lesser extent by the N-type VDCC blocker, ω-conotoxin GVIA (ω-CgTx, 5 μM). On the other hand, nifedipine and ω-CgTx had no effect on K+-induced transmitter secretion. ω-Agatoxin IVA (100 nM), a P/Q-type VDCC blocker, prevents acetylcholine release induced by K+ depolarization but failed to affect MEPP frequency in basal conditions. These results suggest that in the mammalian neuromuscular junction Ca2+ enters nerve terminals through at least three different channels, two of them (L- and N-types) mainly related to spontaneous acetylcholine release and the other (P/Q-type) mostly involved in depolarization-induced neurotransmitter release. Ca2+-binding molecule-related spontaneous release apparently binds Ca2+ very rapidly and would probably be located very close to Ca2+ channels, since the fast Ca2+ chelator (BAPTA-AM) significantly reduced MEPP frequency, whereas EGTA-AM, exhibiting slower kinetics, had a lower effect. The increase in MEPP frequency induced by exposing the preparation to hypertonic solutions was affected by neither external Ca2+concentration nor L-, N-, and P/Q-type VDCC blockers, indicating that extracellular Ca2+ is not necessary to produce hyperosmotic neurosecretion. On the other hand, MEPP frequency was diminished by BAPTA-AM and EGTA-AM to the same extent, supporting the view that hypertonic response is promoted by “bulk” intracellular Ca2+concentration increases.
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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.
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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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Murphy, E., L. Levy, L. R. Berkowitz, E. P. Orringer, S. A. Gabel, and R. E. London. "Nuclear magnetic resonance measurement of cytosolic free calcium levels in human red blood cells." American Journal of Physiology-Cell Physiology 251, no. 4 (October 1, 1986): C496—C504. http://dx.doi.org/10.1152/ajpcell.1986.251.4.c496.
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Red blood cells were loaded with 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (FBAPTA) by incubation with 50 microM of the acetoxymethyl ester (FBAPTA-AM), and cytosolic free Ca2+ was monitored with 19F-nuclear magnetic resonance (NMR). Loading with 50 microM FBAPTA-AM, which results in a final FBAPTA level of approximately 0.5 mM, caused only a 25-30% fall in cell ATP as measured by 31P-NMR when 5 mM pyruvate was present. Leakage of the NMR active Ca2+ indicator, which results from cell lysis, was corrected for with the addition of extracellular Eu3+ ions, extracellular ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA), or washing. With this method, we have found basal levels of cytosolic free Ca2+ averaging 61 +/- 6 nM (means +/- SE, n = 19). When the intracellular level of FBAPTA was varied from 0.1 to 1.0 mM, there was no correlation between the level of cytosolic free Ca2+ and the level of loading with FBAPTA. Addition of 10 microM of the Ca2+ ionophore A23187 with extracellular Ca2+ set at different levels by Ca2+-EGTA buffers caused an increase in cytosolic free Ca2+ as expected. Furthermore, ATP depletion caused a two- to three-fold increase in cytosolic free Ca2+, consistent with inhibition of Ca2+ efflux via that Ca2+-ATPase.
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Hagler, Donald J., and Yukiko Goda. "Properties of Synchronous and Asynchronous Release During Pulse Train Depression in Cultured Hippocampal Neurons." Journal of Neurophysiology 85, no. 6 (June 1, 2001): 2324–34. http://dx.doi.org/10.1152/jn.2001.85.6.2324.
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Neurotransmitter release displays at least two kinetically distinct components in response to a single action potential. The majority of release occurs synchronously with action-potential-triggered Ca2+ influx; however, delayed release—also called asynchronous release—persists for tens of milliseconds following the peak Ca2+ transient. In response to trains of action potentials, synchronous release eventually declines, whereas asynchronous release often progressively increases, an effect that is primarily attributed to the buildup of intracellular Ca2+ during repetitive stimulation. The precise relationship between synchronous and asynchronous release remains unclear at central synapses. To gain better insight into the mechanisms that regulate neurotransmitter release, we systematically characterized the two components of release during repetitive stimulation at excitatory autaptic hippocampal synapses formed in culture. Manipulations that increase the Ca2+ influx triggered by an action potential—elevation of extracellular Ca2+ or bath application of tetraethylammonium (TEA)—accelerated the progressive decrease in synchronous release (peak excitatory postsynaptic current amplitude) and concomitantly increased asynchronous release. When intracellular Ca2+ was buffered by extracellular application of EGTA-AM, initial depression of synchronous release was equal to or greater than control; however, it quickly reached a plateau without further depression. In contrast, asynchronous release was largely abolished in EGTA-AM. The total charge transfer following each pulse—accounting for both synchronous and asynchronous release—reached a steady-state level that was similar between control and EGTA-AM. A portion of the decreased synchronous release in control conditions therefore was matched by a higher level of asynchronous release. We also examined the relative changes in synchronous and asynchronous release during repetitive stimulation under conditions that highly favor asynchronous release by substituting extracellular Ca2+ with Sr2+. Initially, asynchronous release was twofold greater in Sr2+. By the end of the train, the difference was ∼50%; consequently, the total release per pulse during the plateau phase was slightly larger in Sr2+ compared with Ca2+. We thus conclude that while asynchronous release—like synchronous release—is limited by vesicle availability, it may be able to access a slightly larger subset of the readily releasable pool. Our results are consistent with the view that during repetitive stimulation, the elevation of asynchronous release depletes the vesicles immediately available for release, resulting in depression of synchronous release. This implies that both forms of release share a small pool of immediately releasable vesicles, which is being constantly depleted and refilled during repetitive stimulation.
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Hagelstein, Jill, Pauline Schneider, Jasper de Boer, Esther Hulleman, Owen Williams, Rob Pieters, and Ronald W. Stam. "High Expression of the Ca2+-Binding Proteins S100A8 and S100A9 Cause Glucocorticoid Resistance in MLL-Rearranged Infant Acute Lymphoblastic Leukemia." Blood 114, no. 22 (November 20, 2009): 729. http://dx.doi.org/10.1182/blood.v114.22.729.729.
Full textAbstract:
Abstract Abstract 729 MLL-rearranged Acute Lymphoblastic Leukemia (ALL) in infants (i.e. children <1 year of age) represents an aggressive and difficult to treat type of leukemia, displaying cellular resistance to several chemotherapeutics, especially to glucocorticoids like prednisolone. As prednisolone response is highly predictive for clinical outcome, it is of utmost importance to unravel the mechanism underlying resistance to this drug. To gain insights in the prednisolone resistance mechanism, we compared gene expression profiles (Affymetrix HU133plus2) from prednisolone-resistant and prednisolone-sensitive MLL-rearranged infant ALL patients. This gene signature revealed that multiple genes involved in calcium signaling were up-regulated in prednisolone-resistant samples. Most pronounced up-regulation was observed for the S100 protein family members S100A8 and S100A9, which are calcium-binding proteins, that function in a complex. Quantitative RT-PCR (TaqMan) analyses confirmed that S100A8 and S100A9 mRNA expression was ∼100-fold higher in prednisolone-resistant cells compared to patients sensitive to this drug (p=0.008). Glucocorticoids are known to induce apoptosis by releasing Ca2+ from the endoplasmic reticulum (ER) into the cytosol and towards the mitochondria. Within mitochondria, elevated Ca2+ levels induce cytochrome c release, triggering apoptosis. Since S100A8/A9 are capable of binding free cytosolic Ca2+, we hypothesized that over-representation of these cytosolic proteins may prevent Ca2+ to reach the mitochondria and forestall apoptosis. To test our hypothesis, we first co-incubated prednisolone-sensitive MLL-rearranged ALL cells with prednisolone and EGTA or BAPTA-AM. Both agents represent Ca2+chelators and scavenge free cytosolic Ca2+, thereby mimicking the Ca2+binding by S100A8/A9. Flow cytometry analyses showed that both EGTA and BAPTA-AM inhibited the free cytosolic calcium released by prednisolone. Cytotoxicity tests demonstrated that these prednisolone-sensitive cells became more resistant to prednisolone after co-incubation with either EGTA or BAPTA-AM. Next we asked whether enforced over-expression (using retroviral expression vectors) of S100A8 and/or S100A9 in prednisolone-sensitive MLL-rearranged ALL cells could also inhibit free cytosolic Ca2+and induce prednisolone resistance. Indeed, both S100A8 and S100A9 were capable of binding free cytosolic Ca2+ released by prednisolone, accompanied by a 30-40% increase in cell survival when either S100A8 or S100A9 were over-expressed alone. Simultaneous over-expression of both S100A8 and S100A9 almost completely reversed the prednisolone-sensitive into a prednisolone-resistance phenotype. In conclusion, these findings implicate that high expression of S100A8 and S100A9 contributes to prednisolone resistance in MLL-rearranged infant ALL. Disclosures: No relevant conflicts of interest to declare.
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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.
Full textAbstract:
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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Kornyeyev, Dmytro, Mariano Reyes, and Ariel L. Escobar. "Luminal Ca2+ content regulates intracellular Ca2+ release in subepicardial myocytes of intact beating mouse hearts: effect of exogenous buffers." American Journal of Physiology-Heart and Circulatory Physiology 298, no. 6 (June 2010): H2138—H2153. http://dx.doi.org/10.1152/ajpheart.00885.2009.
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Ca+-induced Ca2+ release tightly controls the function of ventricular cardiac myocytes under normal and pathological conditions. Two major factors contributing to the regulation of Ca2+ release are the cytosolic free Ca2+ concentration and sarcoplasmic reticulum (SR) Ca2+ content. We hypothesized that the amount of Ca2+ released from the SR during each heart beat strongly defines the refractoriness of Ca2+ release. To test this hypothesis, EGTA AM, a high-affinity, slow-association rate Ca2+ chelator, was used as a tool to modify luminal SR Ca2+ content. An analysis of the cytosolic and luminal SR Ca2+ dynamics recorded from the epicardial layer of intact mouse hearts indicated that the presence of EGTA reduced the diastolic SR free Ca2+ concentration and fraction of SR Ca2+ depletion during each beat. In addition, this maneuver shortened the refractory period and accelerated the restitution of Ca2+ release. As a consequence of the accelerated restitution, the frequency dependence of Ca2+ alternans was significantly shifted toward higher heart rates, suggesting a role of luminal SR Ca2+ in the genesis of this highly arrhythmogenic phenomenon. Thus, intra-SR Ca2+ dynamics set the refractoriness and frequency dependence of Ca2+ transients in subepicardial ventricular myocytes.
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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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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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Mathes, C., and S. H. Thompson. "The relationship between depletion of intracellular Ca2+ stores and activation of Ca2+ current by muscarinic receptors in neuroblastoma cells." Journal of General Physiology 106, no. 5 (November 1, 1995): 975–93. http://dx.doi.org/10.1085/jgp.106.5.975.
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The relationship between the depletion of IP3-releasable intracellular Ca2+ stores and the activation of Ca(2+)-selective membrane current was determined during the stimulation of M1 muscarinic receptors in N1E-115 neuroblastoma cells. External Ca2+ is required for refilling Ca2+ stores and the voltage-independent, receptor-regulated Ca2+ current represents a significant Ca2+ source for refilling. The time course of Ca2+ store depletion was measured with fura-2 fluorescence imaging, and it was compared with the time course of Ca2+ current activation measured with nystatin patch voltage clamp. At the time of maximum current density (0.18 + .03 pA/pF; n = 48), the Ca2+ content of the IP3-releasable Ca2+ pool is reduced to 39 + 3% (n = 10) of its resting value. Calcium stores deplete rapidly, reaching a minimum Ca2+ content in 15-30 s. The activation of Ca2+ current is delayed by 10-15 s after the beginning of Ca2+ release and continues to gradually increase for nearly 60 s, long after Ca2+ release has peaked and subsided. The delay in the appearance of the current is consistent with the idea that the production and accumulation of a second messenger is the rate-limiting step in current activation. The time course of Ca2+ store depletion was also measured after adding thapsigargin to block intracellular Ca2+ ATPase. After 15 min in thapsigargin, IP3-releasable Ca2+ stores are depleted by > 90% and the Ca2+ current is maximal (0.19 + 0.05 pA/pF; n = 6). Intracellular loading with the Ca2+ buffer EGTA/AM (10 microM; 30 min) depletes IP3-releasable Ca2+ stores by between 25 and 50%, and it activates a voltage-independent inward current with properties similar to the current activated by agonist or thapsigargin. The current density after EGTA/AM loading (0.61 + 0.32 pA/pF; n = 4) is three times greater than the current density in response to agonist or thapsigargin. This could result from partial removal of Ca(2+)-dependent inactivation.
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Takenaka, Katsunobu, Hiromu Yamada, Noboru Sakai, Takashi Ando, Toshihiko Nakashima, Yasuaki Nishimura, Yukio Okano, and Yoshinori Nozawa. "Cytosolic calcium changes in cultured rat aortic smooth-muscle cells induced by oxyhemoglobin." Journal of Neurosurgery 74, no. 4 (April 1991): 620–24. http://dx.doi.org/10.3171/jns.1991.74.4.0620.
Full textAbstract:
✓ To clarify the mechanism of contractive effects in arteries caused by oxyhemoglobin, changes in the concentration of cytosolic calcium ([Ca++]1) before and after exposure to oxyhemoglobin were measured in vitro in cultured vascular smooth-muscle cells obtained from rat aorta. This was accomplished by preloading these cells with a fluorescent intracellular Ca++ probe fura-2/AM. Oxyhemoglobin induced a significant elevation of [Ca++]1 in vascular smooth-muscle cells which was sustained for 10 minutes. This response was completely abolished by chelating extracellular calcium with ethyleneglycol-bis (β-aminoethylether)-N,N′-tetra-acetic acid (EGTA). Oxyhemoglobin induced no accumulation of mass content of inositol 1,4,5-trisphosphate (IP3(1,4,5)). The oxyhemoglobin-induced elevation of [Ca++]1 was not blocked by verapamil, a calcium antagonist. Serotonin induced a rapid, transient increase of [Ca++]1 followed by a sustained elevation above baseline for 5 minutes. Additions of EGTA or verapamil had a small effect on the peak height of serotonin-induced [Ca++]1 elevation, but the [Ca++]1 level declined more quickly to the basal level in treated compared with control cells. These results indicate that oxyhemoglobin-induced [Ca++]1 elevation is caused by the influx of extracellular calcium, which is independent of the verapamil-blocked voltage-gated calcium channel. The long-lasting high elevation of [Ca++]1 caused by oxyhemoglobin suggests that oxyhemoglobin may contribute to the production of abnormal contractions and/or irreversible damage in vascular smooth-muscle cells.
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Teng, Che-Ming, Ya-Fei Kang, Ya-Ling Chang, Feng-Nien Ko, Shu-Chen Yang, and Feng-Lin Hsu. "ADP-mimicking Platelet Aggregation Caused by Rugosin E, an Ellagitannin Isolated from Rosa rugosa Thunb." Thrombosis and Haemostasis 77, no. 03 (1997): 555–61. http://dx.doi.org/10.1055/s-0038-1656005.
Full textAbstract:
SummaryAmong the nine ellagitannins, rugosin E was the most potent platelet aggregating agent with an EC50 of 1.5 ± 0.1 µM in rabbit platelets and 3.2 ±0.1 µM in human platelets. The aggregations caused by rugosin E and ADP were inhibited by EGTA, PGE1, mepacrine, sodium nitroprusside and neomycin, but not by indomethacin, verapamil, TMB-8, BN52021 and GR32191B. Rugosin E-induced thromboxane formation was suppressed by indomethacin, EGTA, PGE,, verapamil, mepacrine, TMB-8 and neomycin. ADP-scavenging agents, such as CP/CPK and apyrase inhibited concentration-dependently ADP (20 εM)-, but not rugosin E (5 εM)-induced platelet aggregation. In thrombin (0.1 U/ml)-treated and degranulated platelets, rugosin E and ADP still caused 63.5 ± 3.0% and 61.2 ± 3.5% of platelet aggregation, respectively. Selective ADP receptor antagonists, ATP and FSBA inhibited rugosin E- and ADP-induced platelet aggregations in a concentration-dependent manner. Both rugosin E and ADP did not induce platelet aggregation in ADP (1 mM)-desensitized platelets. In contrast to ADP, rugosin E did not decrease cAMP formation in washed rabbit platelets. Both rugosin E and ADP did not cause phosphoinositide breakdown in [3H]myo-inositol-labeled rabbit platelets. In fura-2/AM- load platelets, both rugosin E and ADP induced increase in intracellular calcium concentration and these responses were inhibited by ATP and PGEj. All these data suggest that rugosin E may be an ADP receptor agonist in rabbit platelets.
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Somers, S. D., J. E. Weiel, T. A. Hamilton, and D. O. Adams. "Phorbol esters and calcium ionophore can prime murine peritoneal macrophages for tumor cell destruction." Journal of Immunology 136, no. 11 (June 1, 1986): 4199–205. http://dx.doi.org/10.4049/jimmunol.136.11.4199.
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Abstract Murine macrophages from sites of inflammation develop toward tumoricidal competence by exposure to a macrophage-activating factor such as interferon-gamma (IFN-gamma). To explore the biochemical transductional events initiated by IFN-gamma, peritoneal macrophages from C57BL/6J mice elicited by various sterile irritants were treated in vitro with two pharmacologic agents that mimic the action of certain second messengers. Phorbol myristate acetate (PMA) and the ionophore A23187 cooperatively reproduced the ability of IFN-gamma to prime macrophages for tumoricidal function. Neither agent alone was able to prime macrophages. The two agents acted on the macrophages, and target susceptibility to kill was not altered by PMA and A23187. Only active phorbol esters, which are known to bind and stimulate protein kinase C, were able to cooperate with A23187 to induce priming. A cell-permeable synthetic diacylglycerol (sn-1,2-dioctanoyl glycerol) could also prime for cytolysis. In the presence of PMA, A23187, and EGTA, addition of Ca++ was sufficient for priming, whereas the addition of Mg++ was much less efficient. Priming by IFN-gamma, however, was not blocked by EGTA. Efflux of 45Ca++ from preloaded cells was significantly increased by A23187 and by IFN-gamma. Quin-2/AM, an intracellular chelator of Ca++, blocked priming by IFN-gamma. In summary, the data suggest that priming of macrophages for tumoricidal function by IFN-gamma involves, at least in part, alterations in protein kinase C and in levels of intracellular Ca++.
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Kaur, Kuljeet, Jian Yao, Xiaolei Pan, Carolyn Matthews, and Aviv Hassid. "NO decreases phosphorylation of focal adhesion proteins via reduction of Ca in rat aortic smooth muscle cells." American Journal of Physiology-Heart and Circulatory Physiology 274, no. 5 (May 1, 1998): H1613—H1619. http://dx.doi.org/10.1152/ajpheart.1998.274.5.h1613.
Full textAbstract:
Our laboratory has previously reported that the antimitogenic effect of nitric oxide (NO) in primary cultures of rat aortic smooth muscle cells may be attributed to activation of protein tyrosine phosphatase and dephosphorylation of protein phosphotyrosine [G. S. Dhaunsi, C. Matthews, K. Kaur, and A. Hassid. Am. J. Physiol. 272 ( Heart Circ. Physiol. 41): H1342–H1349, 1997]. The goal of the current study was to investigate the role of cytoplasmic Ca in this process and to identify protein substrates that are dephosphorylated by treatment with NO. Treatment of primary rat aortic smooth muscle cell cultures with the NO donor S-nitroso- N-acetylpenicillamine (SNAP) decreased cytoplasmic Ca levels and elicited phosphotyrosine dephosphorylation. Both effects were mimicked by the extracellular and intracellular Ca chelators ethylene glycol-bis(β-aminoethyl ether)- N, N, N′, N′-tetraacetic acid (EGTA) and 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid (BAPTA), respectively, and by the Ca channel blocker nifedipine. Conversely, elevation of cytoplasmic Ca via the use of the Ca ionophore A-23187 or high extracellular K+prevented or attenuated SNAP-induced dephosphorylation. Both BAPTA and nifedipine also decreased DNA synthesis, providing further evidence to link dephosphorylation to antimitogenesis. Two of the proteins dephosphorylated by treatment of cells with NO or EGTA were identified as the focal adhesion proteins, cortactin and paxillin. These results indicate that NO-induced dephosphorylation of protein phosphotyrosine is mediated by reduction of cytoplasmic Ca and suggest that dephosphorylation of focal adhesion proteins may be of relevance to the antimitogenic effect of NO.
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Kotlikoff, M. I. "Potassium currents in canine airway smooth muscle cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 259, no. 6 (December 1, 1990): L384—L395. http://dx.doi.org/10.1152/ajplung.1990.259.6.l384.
Full textAbstract:
The electrical properties of dissociated canine tracheal smooth muscle cells were examined using the whole cell patch-clamp technique. In current clamp mode, current clamp steps did not initiate action potentials but showed clear outward rectification, which was abolished when cells were loaded with Cs+ ions and when tetraethylammonium (TEA+) ions replaced Na+ in the bath solution. In voltage-clamp experiments, depolarizations positive to -45 mV evoked brief voltage-dependent inward Ca2+ currents [Am. J. Physiol. 254 (Cell Physiol. 23): C793-C801, 1988], followed by sustained outward currents, which did not completely inactivate. Outward currents were identified as K+ currents on the basis of the reversal potential of the current and by ion-substitution experiments. The currents were further defined as Ca2(+)-insensitive delayed rectifier currents, since they were unaltered under conditions in which 1) the Ca2+ current was completely blocked by Mn2+ or nifedipine (10 microM); 2) Ba2+ ions were substituted for Ca2+ as the inward current charge carrier; or 3) charybdotoxin (40 nM) or TEA+ (up to 10 mM) were added to the bath. A Ca2(+)-activated potassium [K(Ca)] current was activated by application of methacholine (100 microM), or A23187 (1 microM), under conditions of low Ca2+ buffering capacity in the internal solution [0.3 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N-,N--tetraacetic acid (EGTA)]. The K(Ca) current was blocked by 10 mM TEA+ and was not observed under conditions of high intracellular Ca2+ buffering (11 mM EGTA). These data indicate that canine airway smooth muscle cells contain voltage-dependent delayed rectifier channels that underlie membrane rectification and K(Ca) channels that are activated by agents which release intracellular Ca2+ stores.
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Ido, Masaru, Shinya Kato, Hiroyuki Ogawa, Kenji Hayashi, Yoshihiro Komada, Xao Li Zhang, Minoru Sakurai, and Koji Suzuki. "Ca2+-dependent Activation of the 33-kDa Protein Kinase Transmits Thrombin Receptor Signals in Human Platelets." Thrombosis and Haemostasis 76, no. 03 (1996): 439–43. http://dx.doi.org/10.1055/s-0038-1650596.
Full textAbstract:
SummaryThrombin stimulation induces a dramatic increase in the activity of the 33-kDa serine/threonine kinase (PK33) in human platelets (10). The Arg-Gly-Asp (RGD) peptide, an inhibitor of the thrombin-mediated aggregation of platelets, did not affect the PK33 activation induced by thrombin suggesting that the activation of this kinase occurs independently from platelet aggregation. To identify a potential role of Ca2+ and calmodulin in the regulation of PK33, the effect of several Ca2+/calmodulin inhibitors on the thrombin-induced activation of PK33 was assessed using denaturation/renaturation method. Pretreatment of platelets with EGTA decreased the maximum PK33 activity induced by thrombin. The chelation of both the extra- and the intracellular Ca2+ by EGTA and by acetoxymethyl ester of 5,5′ -dimethyl-bis-(<9-aminophen-oxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM) decreased further the PK33 activation by thrombin. Preincubation of platelets with the anticalmodulin agent, N-(4-aminobutyl)-5-chloro-2-naphtha-lenesulfonamide (W13), inhibited markedly the activation of PK33 by thrombin, whereas the inactive structural analog N-(4-aminobutyl)-2-naphthalenesulfonamide (W12) and the myosin light chain kinase inhibitor 1 -(5-chloronaphthalene-1 -sulfonyl)-1 H-hexahydro-1,4-diaze-pine (ML9) showed very weak inhibitory effects. Treatment of resting platelets with the calcium ionophore, A23187, activated PK33 in a dose-dependent manner; phorbol 12-myristate 13-acetate enhanced this effect. However, the two foregoing agents did not induce similar degree of PK33 activities as thrombin. These results indicate that the activation of PK33 is independent of the formation of the GPIIb/IIIa-fibrinogen complex and that it might be regulated by a Ca2+-dependent pathway.
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Angleson, J. K., and W. J. Betz. "Intraterminal Ca2+ and Spontaneous Transmitter Release at the Frog Neuromuscular Junction." Journal of Neurophysiology 85, no. 1 (January 1, 2001): 287–94. http://dx.doi.org/10.1152/jn.2001.85.1.287.
Full textAbstract:
We investigated the relationship between intraterminal Ca2+concentration ([Ca2+]i) and the frequency of miniature end plate potentials (MEPPs) at the frog neuromuscular junction by use of ratiometric imaging of fura-2-loaded nerve terminals and intracellular recording of MEPPs. Elevation of extracellular [KCl] over the range of 2–20 mM resulted in increases in [Ca2+]i and MEPP frequency. Loading terminals with the fast and slow Ca2+-buffers bis-( o-aminophenoxy)- N,N,N′,N′-tetraacetic acid-acetoxymethyl (BAPTA-AM) and EGTA-AM resulted in equivalent reductions in the KCl-dependent increases in MEPP frequency. The [Ca2+]i dependence of MEPP frequency determined by elevation of [Ca2+]i due to application of 0.1–10 μM ionomycin was similar to that determined when [Ca2+]i was raised by increasing extracellular KCl. Measurements in 10 mM extracellular KCl revealed that application of the phorbol ester phorbol 12 myristate 13-acetetate (PMA) caused an increase in MEPP frequency while the inactive analogue, 4α-PMA, did not. PMA application also caused an increase in [Ca2+]i. The relationship between [Ca2+]i and MEPP frequency in PMA was the same as was determined by the other methods of raising [Ca2+]i. Under all conditions tested, our data revealed a low [Ca2+]i threshold for activation of transmitter release and are consistent with a K d for [Ca2+]i on the order of 1 μM.
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Roberts, Susan R., Martin M. Knight, David A. Lee, and Dan L. Bader. "Mechanical compression influences intracellular Ca2+ signaling in chondrocytes seeded in agarose constructs." Journal of Applied Physiology 90, no. 4 (April 1, 2001): 1385–91. http://dx.doi.org/10.1152/jappl.2001.90.4.1385.
Full textAbstract:
Ca2+ signaling forms part of a possible mechanotransduction pathway by which chondrocytes may alter their metabolism in response to mechanical loading. In this study, a well-characterized model system utilizing bovine articular chondrocytes embedded in 4% agarose constructs was used to investigate the effect of physiological mechanical compressive strain applied after 1 and 3 days in culture. The intracellular Ca2+ concentration was measured by use of the ratiometric Ca2+ indicator indo 1-AM and confocal microscopy. A positive Ca2+ response was defined as a percent increase in Ca2+ ratio above a preset threshold. A significantly greater percentage of cells exhibited a positive Ca2+ response in strained constructs compared with unstrained controls at both time points. In strained constructs, treatment with either Ga3+ or EGTA significantly reduced the number of positive Ca2+ responders compared with untreated controls. These results represent an important step in understanding the physiological role of intracellular Ca2+in chondrocytes under mechanical compression.
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Brailoiu, G. Cristina, Eugen Brailoiu, Raman Parkesh, Antony Galione, Grant C. Churchill, Sandip Patel, and Nae J. Dun. "NAADP-mediated channel ‘chatter’ in neurons of the rat medulla oblongata." Biochemical Journal 419, no. 1 (March 13, 2009): 91–99. http://dx.doi.org/10.1042/bj20081138.
Full textAbstract:
NAADP (nicotinic acid–adenine dinucleotide phosphate) is a potent Ca2+-mobilizing messenger that stimulates Ca2+ release in a variety of cells. NAADP-sensitive Ca2+ channels are thought to reside on acidic Ca2+ stores and to be functionally coupled to IP3 (inositol 1,4,5-trisphosphate) and/or ryanodine receptors located on the endoplasmic reticulum. Whether NAADP-sensitive Ca2+ channels ‘chatter’ to other channels, however, is not clear. In the present study, we have used a cell-permeant NAADP analogue to probe NAADP-mediated responses in rat medulla oblongata neurons. NAADP-AM (NAADP-acetoxymethyl ester) evoked global cytosolic Ca2+ signals in isolated neurons that were reduced in amplitude by removal of external Ca2+, abolished by disruption of acidic compartments and substantially inhibited by blockade of ryanodine receptors. In rat medullary slices, NAADP-AM depolarized neurons from the nucleus ambiguus in the presence of intracellular EGTA, but not of the faster Ca2+ chelator BAPTA [1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid]. Depolarization was also dependent upon extracellular Ca2+, acidic stores and ryanodine receptors. In voltage-clamp mode, NAADP-AM induced an inward current with a reversal potential of approx. 0 mV. The results of the present study reveal the presence of acidic NAADP-sensitive Ca2+ stores in medulla neurons, the mobilization of which results not only in global Ca2+ signals but also in local signals that activate non-selective cation channels on the cell surface resulting in depolarization. Thus NAADP is capable of co-ordinating channels both within the cell interior and at the cell membrane representing a novel mechanism for excitation of central neurons.
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Takenaka, Katsunobu, Hiromu Yamada, Noboru Sakai, Takashi Ando, Toshihiko Nakashima, and Yasuaki Nishimura. "Induction of cytosolic free calcium elevation in rat vascular smooth-muscle cells by cerebrospinal fluid from patients after subarachnoid hemorrhage." Journal of Neurosurgery 75, no. 3 (September 1991): 452–57. http://dx.doi.org/10.3171/jns.1991.75.3.0452.
Full textAbstract:
✓ The purpose of this study was to determine the effects of cerebrospinal fluid (CSF) from patients with subarachnoid hemorrhage (SAH) on cytosolic free calcium in cultured rat vascular smooth-muscle cells using the fluorescent intracellular calcium indicator fura-2/AM. Samples of CSF were collected from 12 patients (seven with and five without vasospasm) on Days 2, 6, 11, and 16 after SAH. Control CSF samples were obtained from five patients 6 to 9 months after they had undergone successful aneurysm surgery following an SAH. All CSF samples in both the non-vasospasm and vasospasm groups, regardless of the day of sampling after the SAH, induced significantly higher transient intracellular calcium elevations when compared to levels induced by control CSF. Furthermore, the addition of 2 mM ethyleneglycol-bis (β-aminoethylether)-N,N'-tetra-acetic acid (EGTA) caused a slight reduction in the peak height in the CSF-induced intracellular calcium rise which declined more rapidly to basal levels than those studied without EGTA. In the non-vasospasm group, the intracellular calcium concentration remained stable after SAH throughout the study period. In contrast, in the vasospasm group, this concentration was highest on Day 2 post-SAH, but sharply decreased on Day 6 and rose again on Day 11. This result correlated with the clinical signs of vasospasm in these patients. These findings indicated that the intracellular calcium elevations induced by CSF obtained after SAH were due to the combination of the influx of extracellular calcium and the mobilization of intracellular calcium from storage sites. The changes in intracellular calcium concentrations in vascular smooth-muscle cells induced by CSF obtained from patients on successive days following SAH suggest that the substances that induce this repeat calcium elevation on Day 11 post-SAH may be the key spasmogens for vasospasm after SAH.
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Velarde, Victoria, Alicia J. Jenkins, Julie Christopher, Timothy J. Lyons, and Ayad A. Jaffa. "Activation of MAPK by modified low-density lipoproteins in vascular smooth muscle cells." Journal of Applied Physiology 91, no. 3 (September 1, 2001): 1412–20. http://dx.doi.org/10.1152/jappl.2001.91.3.1412.
Full textAbstract:
A high concentration of circulating low-density lipoproteins (LDL) is a major risk factor for atherosclerosis. Native LDL and LDL modified by glycation and/or oxidation are increased in diabetic individuals. LDL directly stimulate vascular smooth muscle cell (VSMC) proliferation; however, the mechanisms remain undefined. The extracellular signal-regulated kinase (ERK) pathway mediates changes in cell function and growth. Therefore, we examined the cellular effects of native and modified LDL on ERK phosphorylation in VSMC. Addition of native, mildly modified (oxidized, glycated, glycoxidized) and highly modified (highly oxidized, highly glycoxidized) LDL at 25 μg/ml to rat VSMC for 5 min induced a fivefold increase in ERK phosphorylation. To elucidate the signal transduction pathway by which LDL phosphorylate ERK, we examined the roles of the Ca2+/calmodulin pathway, protein kinase C (PKC), src kinase, and mitogen-activated protein kinase kinase (MEK). Treatment of VSMC with the intracellular Ca2+ chelator EGTA-AM (50 μmol/l) significantly increased ERK phosphorylation induced by native and mildly modified LDL, whereas chelation of extracellular Ca2+ by EGTA (3 mmol/l) significantly reduced LDL-induced ERK phosphorylation. The calmodulin inhibitor N-(6-aminohexyl)-1-naphthalenesulfonamide (40 μmol/l) significantly decreased ERK phosphorylation induced by all types of LDL. Downregulation of PKC with phorbol myristate acetate (5 μmol/l) markedly reduced LDL-induced ERK phosphorylation. Pretreatment of VSMC with a cell-permeable MEK inhibitor (PD-98059, 40 μmol/l) significantly decreased ERK phosphorylation in response to native and modified LDL. These findings indicate that native and mildly and highly modified LDL utilize similar signaling pathways to phosphorylate ERK and implicate a role for Ca2+/calmodulin, PKC, and MEK. These results suggest a potential link between modified LDL, vascular function, and the development of atherosclerosis in diabetes.
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Khundmiri, Syed J., Melissa A. Metzler, Mohamed Ameen, Vishal Amin, Madhavi J. Rane, and Nicholas A. Delamere. "Ouabain induces cell proliferation through calcium-dependent phosphorylation of Akt (protein kinase B) in opossum kidney proximal tubule cells." American Journal of Physiology-Cell Physiology 291, no. 6 (December 2006): C1247—C1257. http://dx.doi.org/10.1152/ajpcell.00593.2005.
Full textAbstract:
Cardiotonic glycosides, like ouabain, inhibit Na+-K+-ATPase. Recent evidence suggests that low molar concentrations of ouabain alter cell growth. Studies were conducted to examine the effect of ouabain on Akt phosphorylation and rate of cell proliferation in opossum kidney (OK) proximal tubule cells. Cells exposed to 10 nM ouabain displayed increased Akt Ser473 phosphorylation, as evidenced by an increase in phospho-Akt Ser473 band density. Ouabain-stimulated Akt Ser473 phosphorylation was inhibited by pretreatment with phosphatidylinositol 3-kinase (PI3K) inhibitors (LY294002 and wortmannin), a PLC inhibitor (edelfosine), and an Akt inhibitor. Moreover, ouabain-mediated Akt Ser473 phosphorylation was suppressed by reduction of extracellular calcium (EGTA) or when intracellular calcium was buffered by BAPTA-AM. An inhibitor of calcium store release (TMB-8) and an inhibitor of calcium entry via store-operated calcium channels ( SKF96365 ) also suppressed ouabain-mediated Akt Ser473 phosphorylation. In fura-2 AM-loaded cells, 10 nM ouabain increased capacitative calcium entry (CCE). Ouabain at 10 nM did not significantly alter baseline cytoplasmic calcium concentration in control cells. However, treatment with 10 nM ouabain caused a significantly higher ATP-mediated calcium store release. After 24 h, 10 nM ouabain increased the rate of cell proliferation. The Akt inhibitor, BAPTA-AM, SKF96365 , and cyclopiazonic acid suppressed the increase in the rate of cell proliferation caused by 10 nM ouabain. Ouabain at 10 nM caused a detectable increase in 86Rb uptake but did not significantly alter Na+-K+-ATPase (ouabain-sensitive pNPPase) activity in crude membranes or cell sodium content. Taken together, the results point to a role for CCE and Akt phosphorylation, in response to low concentrations of ouabain, that increase the rate of cell proliferation without inhibiting Na+-K+-ATPase-mediated ion transport.
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Halseth, Amy E., Robert M. O'Doherty, Richard L. Printz, Deanna P. Bracy, Daryl K. Granner, and David H. Wasserman. "Role of Ca2+ fluctuations in L6 myotubes in the regulation of the hexokinase II gene." Journal of Applied Physiology 88, no. 2 (February 1, 2000): 669–73. http://dx.doi.org/10.1152/jappl.2000.88.2.669.
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Expression of the hexokinase (HK) II gene in skeletal muscle is upregulated by electrically stimulated muscle contraction and moderate-intensity exercise. However, the molecular mechanism by which this occurs is unknown. Alterations in intracellular Ca2+homeostasis accompany contraction and regulate gene expression in contracting skeletal muscle. Therefore, as a first step in understanding the exercise-induced increase in HK II, the ability of Ca2+ to increase HK II mRNA was investigated in cultured skeletal muscle cells, namely L6 myotubes. Exposure of cells to the ionophore A-23187 resulted in an approximately threefold increase in HK II mRNA. Treatment of cells with the extracellular Ca2+chelator EGTA did not alter HK II mRNA, nor was it able to prevent the A-23187-induced increase. Treatment of cells with the intracellular Ca2+ chelator 1,2-bis( o-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) also resulted in an approximately threefold increase in HK II mRNA in the absence of ionophore, which was similar to the increase in HK II mRNA induced by the combination of BAPTA-AM and A-23187. In summary, a rise in intracellular Ca2+ is not necessary for the A-23187-induced increase in HK II mRNA, and increases in HK II mRNA occur in response to treatments that decrease intracellular Ca2+ stores. Depletion of intracellular Ca2+stores may be one mechanism by which muscle contraction increases HK II mRNA.
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Roberts, Diane E., Archibald McNicol, and Ratna Bose. "Mechanism of Collagen Activation in Human Platelets." Journal of Biological Chemistry 279, no. 19 (February 23, 2004): 19421–30. http://dx.doi.org/10.1074/jbc.m308864200.
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The mechanism of collagen-induced human platelet activation was examined using Ca2+, Na+, and the pH-sensitive fluorescent dyes calcium green/fura red, sodium-binding benzofuran isophthalate, and 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Administration of a moderate dose of collagen (10 μg/ml) to human platelets resulted in an increase in [Ca2+]iand platelet aggregation. The majority of this increase in [Ca2+]iresulted from the influx of calcium from the extracellular milieu via the Na+/Ca2+exchanger (NCX) functioning in the reverse mode and was reduced in a dose-dependent manner by the NCX inhibitors 5-(4-chlorobenzyl)-2′,4′-dimethylbenzamil (KD50= 4.7 ± 1.1 μm) and KB-R7943 (KD50= 35.1 ± 4.8 μm). Collagen-induced platelet aggregation was dependent on an increase in [Ca2+]iand could be inhibited by chelation of intra- and extracellular calcium through the administration of 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) and EGTA, respectively, or via the administration of BAPTA-AM to platelets suspended in no-Na+/HEPES buffer. Collagen induced an increase in [Na+]i(23.2 ± 7.6 mm) via the actions of thromboxane A2and, to a lesser extent, of the Na+/H+exchanger. This study demonstrates that the collagen-induced increase in [Ca2+]iis dependent on the concentration of Na+in the extracellular milieu, indicating that the collagen-induced increase in [Na+]icauses the reversal of the NCX, ultimately resulting in an increase in [Ca2+]iand platelet aggregation.
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Liu, Xiaoni, Nana Zhang, Yingjiong Ding, Dongqing Cao, Ying Huang, Xiangjun Chen, Rui Wang, and Ning Lu. "Hydrogen Sulfide Regulates the [Ca2+]iLevel in the Primary Medullary Neurons." Oxidative Medicine and Cellular Longevity 2016 (2016): 1–10. http://dx.doi.org/10.1155/2016/2735347.
Full textAbstract:
In the present study, we attempted to elucidate mechanisms for the regulation of intracellular calcium levels by H2S in primary rat medullary neurons. Our results showed that NaHS significantly increased the level ofCa2+iin rat medullary neurons in a concentration-dependent manner. L-Cysteine and SAM significantly raised the level ofCa2+iin the medullary neurons while HA and/or AOAA produced a reversal effect. In addition, L-cysteine and SAM significantly increased but HA and/or AOAA decreased the production of H2S in the cultured neurons. TheCa2+ielevation induced by H2S was significantly diminished by EGTA-Ca2+-free solutions, and this elevation was also reduced by nifedipine or nimodipine and mibefradil, suggesting the role of L-type and/or T-type Ca2+channels. Moreover, the effect of H2S onCa2+ilevel in neurons was significantly attenuated by BAPTA-AM and thapsigargin, suggesting the source of Ca2+. Therefore, we concluded that both exogenous and endogenous H2S elevatesCa2+ilevel in primarily cultured rat medullary neurons via both increasing calcium influx and mobilizing intracellular Ca2+stores from ER.
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Klepeis, Veronica E., Ann Cornell-Bell, and Vickery Trinkaus-Randall. "Growth factors but not gap junctions play a role in injury-induced Ca2+ waves in epithelial cells." Journal of Cell Science 114, no. 23 (December 1, 2001): 4185–95. http://dx.doi.org/10.1242/jcs.114.23.4185.
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This paper characterizes the early responses of epithelial cells to injury. Ca2+ is an important early messenger that transiently increases in the cytoplasm of cells in response to external stimuli. Its elevation leads to the regulation of signaling pathways responsible for the downstream events important for wound repair, such as cell migration and proliferation. Live cell imaging in combination with confocal laser scanning microscopy of fluo-3 AM loaded cells was performed. We found that mechanical injury in a confluent region of cells creates an elevation in Ca2+ that is immediately initiated at the wound edge and travels as a wave to neighboring cells, with [Ca2+]i returning to background levels within two minutes. Addition of epidermal growth factor (EGF), but not platelet-derived growth factor-BB, resulted in increased [Ca2+]i, and EGF specifically enhanced the amplitude and duration of the injury-induced Ca2+ wave. Propagation of the Ca2+ wave was dependent on intracellular Ca2+ stores, as was demonstrated using both thapsigargin and Ca2+ chelators (EGTA and BAPTA/AM). Injury-induced Ca2+ waves were not mediated via gap junctions, as the gap-junction inhibitors 1-heptanol and 18α-glycyrrhetinic acid did not alter wave propagation, nor did the cells recover in photobleaching experiments. Additional studies also demonstrated that the wave could propagate across an acellular region. The propagation of the injury-induced Ca2+ wave occurs via diffusion of an extracellular mediator, most probably via a nucleotide such as ATP or UTP, that is released upon cell damage. Movies available on-line
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Moore-Dotson, Johnnie M., Justin S. Klein, Reece E. Mazade, and Erika D. Eggers. "Different types of retinal inhibition have distinct neurotransmitter release properties." Journal of Neurophysiology 113, no. 7 (April 2015): 2078–90. http://dx.doi.org/10.1152/jn.00447.2014.
Full textAbstract:
Neurotransmitter release varies between neurons due to differences in presynaptic mechanisms such as Ca2+ sensitivity and timing. Retinal rod bipolar cells respond to brief dim illumination with prolonged glutamate release that is tuned by the differential release of GABA and glycine from amacrine cells in the inner retina. To test if differences among types of GABA and glycine release are due to inherent amacrine cell release properties, we directly activated amacrine cell neurotransmitter release by electrical stimulation. We found that the timing of electrically evoked inhibitory currents was inherently slow and that the timecourse of inhibition from slowest to fastest was GABAC receptors > glycine receptors > GABAA receptors. Deconvolution analysis showed that the distinct timing was due to differences in prolonged GABA and glycine release from amacrine cells. The timecourses of slow glycine release and GABA release onto GABAC receptors were reduced by Ca2+ buffering with EGTA-AM and BAPTA-AM, but faster GABA release on GABAA receptors was not, suggesting that release onto GABAA receptors is tightly coupled to Ca2+. The differential timing of GABA release was detected from spiking amacrine cells and not nonspiking A17 amacrine cells that form a reciprocal synapse with rod bipolar cells. Our results indicate that release from amacrine cells is inherently asynchronous and that the source of nonreciprocal rod bipolar cell inhibition differs between GABA receptors. The slow, differential timecourse of inhibition may be a mechanism to match the prolonged rod bipolar cell glutamate release and provide a way to temporally tune information across retinal pathways.
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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.
Full textAbstract:
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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Tombes, R. M., and G. G. Borisy. "Intracellular free calcium and mitosis in mammalian cells: anaphase onset is calcium modulated, but is not triggered by a brief transient." Journal of Cell Biology 109, no. 2 (August 1, 1989): 627–36. http://dx.doi.org/10.1083/jcb.109.2.627.
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Swiss 3T3 fibroblasts and LLC-PK epithelial cells in prometaphase or metaphase were either injected with fura-2 or loaded with the acetoxymethyl ester derivative of fura-2 (fura-2 AM) and monitored by microspectrofluorimetry. With both methods of loading, we observed two aspects of intracellular free calcium (Cai) metabolism. (a) Most fibroblasts and epithelial cells exhibited a gradual rise from 75 nM in metaphase to 185 nM during cleavage, returning to baseline by early G1. (b) Mitotic Swiss 3T3 cells exhibited rapid transient Cai changes, similar to those previously reported [Poenie, M., J. Alderton, R. Y. Tsien, R. A. Steinhardt. 1985. Nature (Lond.). 315:147-149; Poenie, M., J. Alderton, R. Steinhardt, and R. Tsien. 1986. Science (Wash. DC). 233:886-889; Ratan, R., and M. L. Shelanski. 1988. J. Cell Biol. 107:993]. These Cai transients occurred repetitively, often beginning in metaphase and continuing long after daughter cell formation. Eliminating serum or calcium from the medium abolished the transients, but delayed neither the gradual Cai elevation nor anaphase onset. Co-injection of EGTA or 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) with fura-2 in calcium-free medium, but not in calcium containing medium, blocked both anaphase and the sustained Cai elevation in almost all cases. Blocked cells were rescued by returning calcium to the medium, whereupon Cai slowly but steadily rose as the cell entered anaphase. Spindle microtubules persisted through the EGTA block. Depolymerization of spindle microtubules by nocodazole also reversibly blocked anaphase onset and the sustained Cai elevation, but did not block transients. This study has revealed the following: (a) anaphase in mammalian fibroblasts and epithelial cells is not triggered by brief calcium transients; (b) anaphase is a calcium-modulated event, usually accompanied by a sustained elevation of Cai above 50 nM; (c) the elevation of Cai is dependent upon an intact spindle; and (d) fibroblasts progress through mitosis by drawing upon either intracellular or extracellular sources of calcium.
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Foresta, C., M. Rossato, P. Bordon, and F. Di Virgilio. "Extracellular ATP activates different signalling pathways in rat Sertoli cells." Biochemical Journal 311, no. 1 (October 1, 1995): 269–74. http://dx.doi.org/10.1042/bj3110269.
Full textAbstract:
1. The present study describes effects of extracellular ATP (ATPe) on plasma membrane potential and cytoplasmic Ca2+ concentrations ([Ca2+]i) in rat Sertoli cells. Sertoli cells in suspension were stimulated with ATPe and other nucleotides and ionic changes were monitored utilizing the fluorescent dyes bis-oxonol and fura-2/AM. ATPe induced a prompt plasma membrane depolarization which was dependent on Na+ influx from the extracellular medium, since it was abolished by omission of extracellular Na+. Depolarization was independent of [Ca2+]i rise as it also occurred in the absence of extracellular Ca2+ and after intracellular Ca2+ stores were discharged with thapsigargin. ATPe also stimulated a rapid and biphasic increase in [Ca2+]i: a prompt spike was followed by a prolonged sustained plateau. The initial spike was dependent on Ca2+ release from intracellular stores since it was also present when cells were incubated in EGTA-supplemented Ca(2+)-free medium and was abolished by pretreatment with ionomycin and thapsigargin, agents that discharge intracellular Ca2+ stores. The sustained phase was dependent on Ca2+ influx from the extracellular medium as it was abolished when cells were incubated in EGTA-supplemented Ca(2+)-free medium. Ca2+ influx was due to activation of voltage-operated calcium channels (VOCCs) since it was abolished by the VOCC inhibitors verapamil and nifedipine or incubation in sucrose medium, an experimental condition which precludes plasma membrane depolarization by ATPe. 2. ATPe-induced rises in intracellular Ca2+ concentration and plasma membrane depolarization were reduced by pretreatment with pertussis toxin, suggesting that ATPe-activated transduction mechanisms are in part under the control of pertussis toxin-sensitive G-proteins. These data show that Sertoli cells possess P2-purinergic receptor subtypes coupled to influx of Na+ and release of Ca2+ from intracellular stores and provide evidence for an activation of different pathways by extracellular ATPe. Activation of these receptors induces Na+ influx that causes a rapid plasma membrane depolarization. Furthermore, ATPe also triggers Ca2+ release from intracellular stores and Ca2+ influx from extracellular space via dihydropyridine-sensitive VOCCs.
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Yi, Fu-Xian, Andrew Y. Zhang, William B. Campbell, Ai-Ping Zou, Cornelis van Breemen, and Pin-Lan Li. "Simultaneous in situ monitoring of intracellular Ca2+ and NO in endothelium of coronary arteries." American Journal of Physiology-Heart and Circulatory Physiology 283, no. 6 (December 1, 2002): H2725—H2732. http://dx.doi.org/10.1152/ajpheart.00428.2002.
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We developed an in situ assay system to simultaneously monitor intracellular Ca2+concentration ([Ca2+]i, fura 2 as indicator) and nitric oxide (NO) levels [4,5-diaminofluorescein as probe] in the intact endothelium of small bovine coronary arteries by using a fluorescent microscopic imaging technique with high-speed wavelength switching. Bradykinin (BK; 1 μM) stimulated a rapid increase in [Ca2+]i followed by an increase in NO production in the endothelial cells. The protein tyrosine phosphatase inhibitor phenylarsine oxide (PAO; 10 μM) induced a gradual, small increase in [Ca2+]i and a slow increase in intracellular NO levels. Removal of extracellular Ca2+ and depletion of Ca2+ stores completely blocked BK-induced increase in NO production but had no effect on PAO-induced NO production. However, a further reduction of [Ca2+]i by application of BAPTA-AM or EGTA with ionomycin abolished the PAO-induced NO increase. These results indicate that a simultaneous monitoring of [Ca2+]i and intracellular NO production in the intact endothelium is a powerful tool to study Ca2+-dependent regulation of endothelial nitric oxide synthase, which provides the first direct evidence for a permissive role of Ca2+ in tyrosine phosphorylation-induced NO production.
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Sun, Suxia, Wenjun Li, He Zhang, Longying Zha, Yong Xue, Xianbo Wu, and Fei Zou. "Requirement for store-operated calcium entry in sodium butyrate-induced apoptosis in human colon cancer cells." Bioscience Reports 32, no. 1 (October 10, 2011): 83–90. http://dx.doi.org/10.1042/bsr20110062.
Full textAbstract:
The SOCE (store-operated Ca2+ entry) pathway plays a key role in both normal cells and cancerous cells. However, its molecular mechanism remains a long-lasting puzzle of Ca2+ signalling. In this paper, we provide evidence that butyric acid, a dietary fibre-derived short-chain fatty acid, induces apoptosis of colon cancer cells via SOCE signalling networks. We found that sodium butyrate (NaB) induces Ca2+ release from endoplasmic reticulum, which in turn causes extracellular Ca2+ influx in HCT-116 cells. The Ca2+ release and influx are important, because the addition of chelators, EGTA or BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid tetrakis(acetoxymethyl ester)] respectively blocked NaB-induced apoptosis. Furthermore, down-regulation of STIM1 (stromal interaction molecule 1) by RNA interference or pharmacological blockade of the SOCC (store-operated Ca2+ channel) by 2-APB (2-aminoethoxydiphenyl borate) or SKF-96365 inhibited NaB-induced extracellular Ca2+ influx and apoptosis in HCT-116 cells. Thus we conclude that NaB triggers colon cancer cell apoptosis in an SOCE-dependent manner. This finding provides new insights into how butyric acid suppresses colon carcinogenesis.
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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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Kapur, Nidhi, Gregory A. Mignery, and Kathrin Banach. "Cell cycle-dependent calcium oscillations in mouse embryonic stem cells." American Journal of Physiology-Cell Physiology 292, no. 4 (April 2007): C1510—C1518. http://dx.doi.org/10.1152/ajpcell.00181.2006.
Full textAbstract:
During cell cycle progression, somatic cells exhibit different patterns of intracellular Ca2+signals during the G0phase, the transition from G1to S, and from G2to M. Because pluripotent embryonic stem (ES) cells progress through cell cycle without the gap phases G1and G2, we aimed to determine whether mouse ES (mES) cells still exhibit characteristic changes of intracellular Ca2+concentration during cell cycle progression. With confocal imaging of the Ca2+-sensitive dye fluo-4 AM, we identified that undifferentiated mES cells exhibit spontaneous Ca2+oscillations. In control cultures where 50.4% of the cells reside in the S phase of the cell cycle, oscillations appeared in 36% of the cells within a colony. Oscillations were not initiated by Ca2+influx but depended on inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+release and the refilling of intracellular stores by a store-operated Ca2+influx (SOC) mechanism. Using cell cycle synchronization, we determined that Ca2+oscillations were confined to the G1/S phase (∼70% oscillating cells vs. G2/M with ∼15% oscillating cells) of the cell cycle. ATP induced Ca2+oscillations, and activation of SOC could be induced in G1/S and G2/M synchronized cells. Intracellular Ca2+stores were not depleted, and all three IP3receptor isoforms were present throughout the cell cycle. Cell cycle analysis after EGTA, BAPTA-AM, 2-aminoethoxydiphenyl borate, thapsigargin, or U-73122 treatment emphasized that IP3-mediated Ca2+release is necessary for cell cycle progression through G1/S. Because the IP3receptor sensitizer thimerosal induced Ca2+oscillations only in G1/S, we propose that changes in IP3receptor sensitivity or basal levels of IP3could be the basis for the G1/S-confined Ca2+oscillations.
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Eickelberg, Oliver, John Geibel, Frank Seebach, Gerhard Giebisch, and Michael Kashgarian. "K+-induced HSP-72 expression is mediated via rapid Ca2+ influx in renal epithelial cells." American Journal of Physiology-Renal Physiology 281, no. 2 (August 1, 2001): F280—F287. http://dx.doi.org/10.1152/ajprenal.2001.281.2.f280.
Full textAbstract:
Pathophysiological stimuli, including hypoxia, lead to K+ efflux from the intracellular lumen to the extracellular space, thereby increasing local tissue K+ concentrations and depolarizing resident cells. In this study, we investigated the effects of increased extracellular K+ concentrations ([K+]e) on heat shock protein (HSP) expression in the porcine proximal tubule epithelial cell line LLC-PK1. We analyzed HSP-25, HSP-72, HSC-73, and HSP-90 protein expression by Western blot analyses and HSP-72 promoter activity by luciferase reporter gene assays using the proximal 1,440 bp of the HSP-72 promoter. Elevating [K+]e from 20 to 50 mM increased HSP-72 protein expression and promoter activity but did not affect HSP-25, HSC-73, or HSP-90 levels. Addition of identical concentrations of sodium chloride did not increase HSP-72 expression to a similar amount. The Ca2+ channel blocker diltiazem and the Ca2+-specific chelator EGTA-AM abolished high [K+]e-induced HSP-72 expression by 69.7 and 75.2%, respectively, indicating that the transcriptional induction of HSP-72 involves Ca2+ influx. As measured by confocal microscopy using the Ca2+ dye fluo 3-AM, we also observed a rapid increase of intracellular Ca2+ concentration as early as 30 s after placing LLC-PK1 cells in high [K+]e. We further analyzed whether Ca2+ influx was necessary for induction of HSP-72 expression by high [K+]e using Ca2+-free medium. Here, induction of HSP-72 in response to high [K+]e was completely abolished. Our data thus demonstrate activation of a protective cellular response to ionic stress, e.g., elevated K+ concentrations, by specifically increasing protein levels of HSP-72.
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Peeters, G. A., V. Hlady, J. H. Bridge, and W. H. Barry. "Simultaneous measurement of calcium transients and motion in cultured heart cells." American Journal of Physiology-Heart and Circulatory Physiology 253, no. 6 (December 1, 1987): H1400—H1408. http://dx.doi.org/10.1152/ajpheart.1987.253.6.h1400.
Full textAbstract:
The fluorescent Ca2+ probe indo-1 is a new intracellular Ca2+ concentration [( Ca2+]i) indicator that may be suitable for measurement of [Ca2+]i transients in intact heart cells. We exposed spontaneously contracting cultured chick embryo ventricular cells (37 degrees C) to the membrane-permeable indo-1-acetoxymethyl ester (indo-1 AM). Indo-1 loading was associated with a decrease in the amplitude of contraction measured with a video motion detector, but contractility returned to control levels during a subsequent 30-min wash. Analysis of emission spectra of dye obtained by digitonin permeabilization of cells loaded in indo-1 AM showed that the active intracellular dye was not pure indo-1 but probably includes partially deesterified molecules. With the use of an inverted X40 objective epifluorescence system, washed cells containing indo-1 were excited at 360 nm, and fluorescence intensity was measured at 410 nm (increases with increasing [Ca2+]) and 480 nm (decreases with increasing [Ca2+]). Calibration of the [Ca2+]i signals, reflected by the ratio of 410 to 480 nm fluorescence, was achieved by use of ethylen-glycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA)-Ca2+ buffered solutions containing the nonfluorescent Ca2+ ionophore Bromo-A23187. Average end-diastolic and peak-systolic [Ca2+]i were 328 +/- 32 and 813 +/- 72 nM (means +/- SE, n = 8). The onset of the [Ca2+]i transient preceded motion by 27 +/- 5 ms (means +/- SE, n = 4), but generally resembled the motion signals in contour. These findings indicate that indo-1 may be used to detect [Ca2+]i transients in isolated ventricular cells without causing significant alterations in mechanical performance.
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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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Li, Yuan, and Madhu B. Anand-Srivastava. "Implication of multiple signaling pathways in the regulation of angiotensin II induced enhanced expression of Giα proteins in vascular smooth muscle cells." Canadian Journal of Physiology and Pharmacology 90, no. 8 (August 2012): 1105–16. http://dx.doi.org/10.1139/y2012-042.
Full textAbstract:
We have previously shown that A10 vascular smooth muscle cells (VSMC) exposed to angiotensin II (Ang II) exhibited overexpression of Giα proteins. In the present study, we examined the involvement of different signaling pathways in regulating Ang II induced enhanced expression of Giα proteins in VSMC by using pharmacological inhibitors. Ang II induced increased expression of Giα proteins in A10 VSMC was markedly attenuated by actinomycin D, losartan (an AT1 receptor antagonist), dibutyryl cAMP, phospholipase C (PLC) inhibitor U73122, protein kinase C (PKC) inhibitors staurosporine and GP109203X, but not by PD123319 (an AT2 receptor antagonist). In addition, BAPTA-AM and TMB-8 (chelators of intracellular Ca2+); and nifedipine (a blocker of L-type Ca2+ channels) significantly inhibited Ang II induced enhanced expression of Giα proteins. On the other hand, extracellular Ca2+ chelation using EGTA did not affect the Ang II evoked enhanced levels of Giα proteins. Furthermore, pretreatment of A10 VSMC with calmidazolium (an inhibitor of calmodulin), or KN93 (an inhibitor of CaM kinase), or genistein (an inhibitor of protein tyrosine kinase, PTK), also attenuated the increased levels of Giα proteins induced by Ang II. These results suggest that Ang II induced enhanced expression of Giα proteins may be regulated by different signaling pathways through AT1 receptors in A10 VSMC.