The alteration of mitotic events by ionophore A23187 and carbonyl cyanide n-chlorophenylhydrazone

1985 ◽  
Vol 75 (1) ◽  
pp. 347-355
Author(s):  
M.L. Ziegler ◽  
J.E. Sisken ◽  
S. Vedbrat
Keyword(s):  
Oxidative Phosphorylation ◽  
Intracellular Calcium ◽  
Calcium Ions ◽  
Hela Cells ◽  
Cytosolic Calcium ◽  
Ionophore A23187 ◽  
Chromosome Movement ◽  
Low Concentrations ◽  
Carbonyl Cyanide ◽  
Stimulation Of

A large quantity of published work indicates that calcium ions may be involved in the regulation of mitotic events and recent reports suggest that the onset of chromosome movement is dependent upon a transient increase in free cytosolic calcium ions. In this paper we examine the effects of two agents known to perturb intracellular calcium pools on mitosis in HeLa cells. These were the calcium-selective ionophore A23187 and carbonyl cyanide n-chlorophenylhydrazone (CCCP), which is a protonophoric inhibitor of oxidative phosphorylation. Owing to a stimulation of glycolysis, the latter agent does not decrease intracellular ATP in HeLa but does cause mitochondria to release calcium ions. Our data show that, at low concentrations, both agents prolong metaphase but differ in their effects on anaphase and cytokinesis. Studies with chlorotetracycline, a commonly used probe for membrane-associated calcium, verify that these agents do affect calcium pools under the conditions of our experiments. The data presented are consistent with the idea that increased cytosolic calcium levels can directly or indirectly affect mitotic events but, contrary to other suggestions, cause a prolongation of metaphase, i.e. they delay the onset of chromosome movement.

scholarly journals Rapid ultrastructural changes in the dense tubular system following platelet activation

Blood ◽  
1992 ◽  
Vol 80 (3) ◽  
pp. 718-723 ◽  
Author(s):  
L Ebbeling ◽  
C Robertson ◽  
A McNicol ◽  
JM Gerrard
Keyword(s):  
Intracellular Calcium ◽  
Platelet Activation ◽  
Calcium Ionophore ◽  
Cytosolic Calcium ◽  
Calcium Ionophore A23187 ◽  
Ultrastructural Changes ◽  
Ionophore A23187 ◽  
Tubular System ◽  
Protein Kinase C Activators ◽  
Morphologic Changes

Abstract The dense tubular system (DTS) functions to regulate platelet activation by sequestering or releasing calcium, similar to the sarcotubules of skeletal muscle. In resting platelets, the DTS exists as thin elongated membranes. Within 10 seconds of the addition of thrombin, platelets show a major ultrastructural change in their DTS: from the thin elongated form to a rounded vesicular form. These morphologic changes were demonstrated with two different stains and two different fixation methods. Platelets exposed to the calcium ionophore A23187 showed the same ultrastructural changes in the DTS. In contrast, the DTS remains in a thin elongated form when platelets are stimulated by the protein kinase C activators phorbol 12-myristate 13-acetate (PMA) and oleoylacetylglycerol (OAG). These morphologic changes may be related to the discharge of calcium from the DTS because this is stimulated by thrombin and A23187, but not by PMA. Preincubation of the platelets with the intracellular calcium chelator 5,5′-dimethyl-bis-(0- aminophenoxy)-ethane-N,N,N′,N tetra acetic acid (BAPTA) largely prevented both the thrombin-induced rise in intracellular calcium and the changes in DTS morphology, suggesting that the changes in DTS morphology are secondary to the increase in cytosolic calcium. The results provide a morphologic correlate to existing biochemical evidence showing that the DTS is involved early during paltelet activation.

scholarly journals Relationship between hormonal activation of phosphatidylinositol hydrolysis, fluid secretion and calcium flux in the blowfly salivary gland

1979 ◽  
Vol 178 (1) ◽  
pp. 45-58 ◽  
Author(s):  
J N Fain ◽  
M J Berridge
Keyword(s):  
Salivary Gland ◽  
Fluid Secretion ◽  
Salivary Secretion ◽  
Calcium Flux ◽  
Ionophore A23187 ◽  
Bivalent Cation ◽  
Inositol 1 ◽  
Low Concentrations ◽  
Hormonal Activation ◽  
Stimulation Of

The addition of 5-hydroxytryptamine to the isolated blowfly salivary gland stimulates fluid secretion, transepithelial calcium transport and the breakdown of 32P- or 3H-labelled phosphatidylinositol The breakdown of [32P]phosphatidylcholine and [32P]-phosphatidylethanolamine was not stimulated by 5-hydroxytryptamine. In salivary glands incubated with myo-[2-3H]inositol for 1–3 h, more than 95% of the label retained by the tissue was in the form of phosphatidylinositol. The addition of 5-hydroxytryptamine resulted in an increase in the accumulation of label in intracellular inositol 1:2-cyclic phosphate, inositol 1-phosphate and free inositol along with an increase in the release of [3H]inositol to the medium and saliva. The release of [3H]inositol to the medium served as a sensitive indicator of phosphatidylinositol breakdown. The release of [3H]inositol was not increased by cyclic AMP or the bivalent-cation ionophore A23187 under conditions in which salivary secretion was accelerated. The stimulation of fluid secretion by low concentrations of 5-hydroxytryptamine was potentiated by 3-isobutyl-1-methylxanthine, which had no effect on inositol release. The stimulation of fluid secretion by 5-hydroxytryptamine was greatly reduced in calcium-free buffer, but the breakdown of phosphatidylinositol continued at the same rate in the absence of calcium. These results support the hypothesis that breakdown of phosphatidylinositol by 5-hydroxytryptamine is involved in the gating of calcium.

scholarly journals Adenosine 3':5'-monophosphate content and actions in the division cycle of synchronized HeLa cells.

1976 ◽  
Vol 71 (2) ◽  
pp. 515-534 ◽  
Author(s):  
C E Zeilig ◽  
R A Johnson ◽  
E W Sutherland ◽  
D L Friedman
Keyword(s):  
Cell Cycle ◽  
Cyclic Amp ◽  
Hela Cells ◽  
S Phase ◽  
Intracellular Camp ◽  
Specific Effects ◽  
Low Concentrations ◽  
High Concentrations ◽  
Camp Levels ◽  
Stimulation Of

The involvement of adenosine 3':5'-monophosphate (cAMP) in the regulation of the cell cycle was studied by determining intracellular fluctuations in cAMP levels in synchronized HeLa cells and by testing the effects of experimentally altered levels on cell cycle traverse. Cyclic AMP levels were lowest during mitosis and were highest during late G-1 or early S phase. These findings were supported by results obtained when cells were accumulated at these points with Colcemid or high levels of thymidine. Additional fluctuations in cAMP levels were observed during S phase. Two specific effects of cAMP on cell cycle traverse were found. Elevation of cAMP levels in S phase or G-2 caused arrest of cells in G-2 for as long as 10 h and lengthened M. However, once cells reached metaphase, elevation of cAMP accelerated the completion of mitosis. Stimulation of mitosis was also observed after addition of CaCl2. The specificity of the effects of cAMP was verified by demonstrating that: (a) intracellular cAMP was increased after exposure to methylisobutylxanthine (MIX) before any observed effects on cycle traverse; (b) submaximal concentrations of MIX potentiated the effects of isoproterenol; and (c) effects of MIX and isoproterenol were mimicked by 8-Br-cAMP. MIX at high concentrations inhibited G-1 traverse, but this effect did not appear to be mediated by cAMP. Isoproterenol slightly stimulated G-1 traverse and partially prevented the MIX-induced delay. Moreover, low concentrations of 8-Br-cAMP (0.10-100 muM) stimulated G-1 traverse, whereas high concentrations (1 mM) inhibited. Both of these effects were also observed with the control, Br-5'-AMP, at 10-fold lower concentrations.

Intracellular Calcium Ions Modulate Permeability of the Peritoneal Mesothelium In Vitro

1985 ◽  
Vol 5 (2) ◽  
pp. 105-108 ◽  
Author(s):  
Andrzej Breborowicz ◽  
Jan Knapowski ◽  
Grzegorz Breborowicz
Keyword(s):  
Epithelial Cells ◽  
Intracellular Calcium ◽  
Local Anesthetic ◽  
Calcium Ions ◽  
Calcium Transport ◽  
Calcium Ionophore ◽  
Ionophore A23187 ◽  
Peritoneal Mesothelium ◽  
Vitro Effect

The authors studied the in vitro effect of a calcium-channel-blocker -verapamil, local anesthetic -bupivacaine and a calcium ionophore -A23187 on the permeability of the monolayer mesothelium from the rabbit's mesentery. Verapamil and bupivacaine increased transmesothelial flux of calcium while A23187 increased calcium transport only transiently. Verapamil augmented the permeability of the mesothelium to inulin and urea. However A23187 decreased the transmesothelial flux of inulin only whereas it increased the transport of urea. From this study we have concluded, that intracellular calcium may determine the permeability of the peritoneal mesothelium. Various important biological processes like muscle contraction, secretion of transmitters and hormones, and control of epithelial permability seem to be dependent on intracellular calcium-ion activity (1–4). Our previous paper suggested that a local anesthetic, bupivacaine, produces its effect on the peritoneal mesothelium through interaction with the cytoskeleton of the epithelial cells, and with the mem brane's calcium transport (5). In addition Palant et al found that the permeability of another leaky epithelium, that from the Necturus gallbladder, depends on the extracellular calcium concentrations and may be altered by drugs, which interfere with the entrance of these ions into the epithelial cells (6). Therefore, we decided to study the role that calcium ions may play in the transport of solutes across the peritoneal mesothelium in vitro.

A role for intracellular calcium and calmodulin in the release of triiodothyronine from human thyroid-cell monolayer cultures

1984 ◽  
Vol 4 (8) ◽  
pp. 695-702 ◽  
Author(s):  
C. A. Ollis ◽  
R. Davies ◽  
D. S. Munro ◽  
S. Tomlinson
Keyword(s):  
Intracellular Calcium ◽  
Cell Monolayer ◽  
Calcium Ionophore ◽  
Cytosolic Calcium ◽  
Human Thyroid ◽  
Free Calcium ◽  
Thyroid Cell ◽  
Thyroid Cells ◽  
Calmodulin Antagonist ◽  
Low Concentrations

Human thyroid cells in monolayer responded to acute stimulation by TSH with an increase in the secretion of T3. This process appeared to be dependent on a rise in the cytosolic calcium concentration since the antagonist of intraceliular calcium mobilization, TMB-8, was found to inhibit the release of T3 in response to TSH. The importance of intracellular calcium was further shown using the agent veratridine which increases the free calcium level within cells; veratridine potentiated the stimulation of T3 secretion by TSH and itself stimulated the release of T3 to a level higher than that seen in the presence of TSH alone. The calcium ionophore A23197 produced a biphasic effect on T3 secretion from human thyroid monolayers; at low concentrations, A23187 caused a decrease in both unstimulated and TSH-stimulated T3 secretion but above a concentration of 1 μM, T3 secretion was increased. The calmodulin antagonist W7 was found to inhibit T3 release in response to TSH, indicating a role for calmodulin in mediating the effects of intracellular calcium on T3 secretion.

scholarly journals Peroxisomes contribute to intracellular calcium dynamics

2020 ◽  
Author(s):  
Yelena Sargsyan ◽  
Uta Bickmeyer ◽  
Katrin Streckfuss-Bömeke ◽  
Ivan Bogeski ◽  
Sven Thoms
Keyword(s):  
Intracellular Calcium ◽  
Hela Cells ◽  
Calcium Influx ◽  
Induced Pluripotent Stem Cell ◽  
Cytosolic Calcium ◽  
Neonatal Rat ◽  
Calcium Handling ◽  
Calcium Stores ◽  
Calcium Dynamics ◽  
Rat Cardiomyocytes

AbstractPeroxisomes communicate with other cellular compartments by transfer of various metabolites. However, whether peroxisomes are sites for calcium handling and exchange has remained contentious. Here we generated sensors for assessment of peroxisomal calcium and applied them for single cell-based calcium imaging in HeLa cells and cardiomyocytes. We found that peroxisomes in HeLa cells take up calcium upon depletion of intracellular calcium stores and upon calcium influx across the plasma membrane. Further, we show that peroxisomes of neonatal rat cardiomyocytes and human induced pluripotent stem cell-derived cardiomyocytes can take up calcium in a controlled manner. Our results indicate that peroxisomal and cytosolic calcium signals are tightly interconnected. Hence, peroxisomes may play an important role in shaping cellular calcium dynamics by serving as buffers or sources of intracellular calcium.

Identification of lactate as a driving force for prostanoid transport by prostaglandin transporter PGT

2002 ◽  
Vol 282 (6) ◽  
pp. F1097-F1102 ◽  
Author(s):  
Brenda S. Chan ◽  
Shinichi Endo ◽  
Naoaki Kanai ◽  
Victor L. Schuster
Keyword(s):  
Carbon Source ◽  
Oxidative Phosphorylation ◽  
Hela Cells ◽  
Driving Force ◽  
Fold Increase ◽  
Lactate Exchange ◽  
Dose Dependent ◽  
Stimulation Of

We previously characterized the prostaglandin (PG) transporter PGT as an exchanger in which [3H]PGE2 influx is coupled to the efflux of a countersubstrate. Here, we cultured HeLa cells that stably expressed human PGT under conditions known to favor glycolysis (glucose as a carbon source) or oxidative phosphorylation (glutamine as a carbon source) and studied the effect on PGT-mediated [3H]PGE2 influx. PGT-expressing cells grown in glutamine exhibited a 2- to 4-fold increase in [3H]PGE2 influx compared with the antisense control, whereas cells grown in glucose exhibited a 14-fold increase. In the presence of 10 vs. 25 mM glucose during the uptake, there was a dose-dependent increment in [3H]PGE2 influx. Cis inhibition of [3H]PGE2 influx occurred with lactate at physiological concentrations (apparent K m = 48 ± 12 mM). Preloading with lactate caused a dose-dependent trans stimulation of PGT-mediated [3H]PGE2 uptake, and external lactate caused trans stimulation of PGT-mediated [3H]PGE2 release. Together, these data are consistent with PGT-mediated PG-lactate exchange. Cells engaged in glycolysis would then be poised energetically for prostanoid uptake by means of PGT.

scholarly journals The role of calcium ions in the process of acetyltransferase activation during the formation of platelet-activating factor (PAF-acether)

1984 ◽  
Vol 219 (2) ◽  
pp. 419-424 ◽  
Author(s):  
J Gómez-Cambronero ◽  
P Iñarrea ◽  
F Alonso ◽  
M Sánchez Crespo
Keyword(s):  
Intracellular Calcium ◽  
Significant Role ◽  
Calcium Ions ◽  
Calcium Transport ◽  
Platelet Activating Factor ◽  
Peritoneal Macrophages ◽  
Threshold Concentration ◽  
Ionophore A23187 ◽  
Different Doses

The role of Ca2+ in the activation of the enzyme lyso-(platelet-activating factor): acetyl-CoA acetyltransferase was studied in rat peritoneal macrophages in response to complement-coated zymosan particles and ionophore A23187. By using Ca2+-containing buffers, a threshold concentration of extracellular Ca2+ above 1 microM was found to be necessary to observe the activation of the enzyme in response to zymosan. By contrast, a significant role of intracellular Ca2+ in this process could be ruled out, since the putative intracellular calcium-transport antagonist TMB-8 [8-(NN-diethylamino)octyl-3,4,5-trimethoxybenzoate] did not inhibit the activation of the acetyltransferase induced by zymosan in the presence of extracellular Ca+. The link between acetyltransferase activation and extracellular Ca2+ transport was studied by measuring Ca2+ uptake in response to the stimuli. Zymosan particles induced a rapid increment in cell-associated Ca2+ which correlated well with the extent of acetyltransferase activation (r = 0.91) and with the release of platelet-activating factor (r = 0.95) in response to different doses of zymosan. Cellular Ca2+ efflux in response to zymosan particles was also measured and found to be increased, as compared with controls, when the activation of the acetyltransferase declined. In short, the data suggest that the entry of extracellular Ca2+ into the cell is a crucial event in the activation of acetyltransferase and, thereby, in the formation of platelet-activating factor in rat peritoneal macrophages.

Carotid body O2 chemoreception and mitochondrial oxidative phosphorylation

1981 ◽  
Vol 51 (2) ◽  
pp. 438-446 ◽  
Author(s):  
E. Mulligan ◽  
S. Lahiri ◽  
B. T. Storey
Keyword(s):  
Oxidative Phosphorylation ◽  
Carotid Body ◽  
High Energy ◽  
Metabolic Inhibitors ◽  
Antimycin A ◽  
Mitochondrial Oxidative Phosphorylation ◽  
High Energy Phosphate ◽  
Afferent Fibers ◽  
Carbonyl Cyanide ◽  
Stimulation Of

The effect on carotid chemoreceptor afferents of oligomycin, an inhibitor of mitochondrial oxidative phosphorylation that does not affect energy conservation, was studied in 20 cats that were anesthetized, paralyzed, and artificially ventilated. Responses of single or a few chemoreceptor afferents to changes in arterial O2 tension (PaO2) at constant arterial CO2 tension were recorded. In addition, responses to nicotine, cyanide, and antimycin A or carbonyl cyanide p-tri-fluoromethoxyphenylhydrazone (FCCP) were tested in normoxia. Oligomycin (50-500 microgram) was administered by close intra-arterial injection, and the same tests were repeated at timed intervals. Initially, oligomycin caused vigorous stimulation of carotid chemoreceptor activity. Subsequently, although the afferent fibers were still active and could be vigorously stimulated by nicotine, they no longer responded to changes in PaO2 or to doses of cyanide, antimycin A, or FCCP. These results separate stimulation of chemoreceptor afferents by hypoxia and metabolic inhibitors and uncouplers from that by nicotine and suggest that intact oxidative phosphorylation, required for maintenance of the intracellular high-energy phosphate levels, forms the basis of O2 chemoreception in the carotid body.

scholarly journals Regulation of Arachidonic Acid Production by Intracellular Calcium in Parathyroid Cells: Effect of Extracellular Phosphate

2002 ◽  
Vol 13 (3) ◽  
pp. 693-698 ◽  
Author(s):  
Yolanda Almadén ◽  
Antonio Canalejo ◽  
Evaristo Ballesteros ◽  
Gracia Añón ◽  
Sagrario Cañadillas ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Intracellular Calcium ◽  
Intracellular Signaling ◽  
Calcium Level ◽  
Parathyroid Tissue ◽  
Ionophore A23187 ◽  
Calcium Receptor ◽  
Intracellular Calcium Level ◽  
High Phosphate ◽  
Stimulation Of

ABSTRACT. The action of extracellular calcium on the calcium receptor in parathyroid cells results in activation of phospholipase C (PLC), PLD, and PLA2. The PLA2-arachidonic acid (AA) intracellular signaling pathway mediates inhibition of parathyroid hormone (PTH) secretion. In addition, stimulation of the calcium receptor produces increases in intracellular calcium levels. It was demonstrated that high extracellular phosphate levels reduce the production of AA, a mechanism by which phosphate may stimulate PTH secretion. The objective was to determine, in parathyroid tissue, whether AA production is stimulated by increases in intracellular calcium levels and to investigate whether the decreased AA production induced by high extracellular phosphate levels could be modified by increases in intracellular calcium levels. Experiments were performed in vitro using parathyroid tissue. The intracellular calcium level was increased by incubation with an ionophore (A23187), which increases calcium influx across the cell membrane, or thapsigargin, which releases calcium from intracellular stores. The phosphate concentration in the medium was normal (1 mM) or high (4 mM). The response to calcium was evaluated by incubation with 0.6 or 1.35 mM calcium concentrations. AA production by parathyroid tissue was measured by gas chromatography. In parathyroid tissue incubated with either a calcium ionophore or thapsigargin, there was an increase in AA production, together with inhibition of PTH secretion, suggesting that PLA2 is activated by the elevation in intracellular calcium levels. Therefore, the effect of intracellular calcium level elevation on AA production in the presence of high extracellular phosphate levels was evaluated. The results demonstrate that, despite high phosphate levels in the medium, both the ionophore and thapsigargin were capable of inducing a marked increase in AA production, which was associated with a decrease in PTH secretion. In conclusion, in parathyroid tissue, AA levels can be regulated by an ionophore and thapsigargin, both of which increase cytosolic calcium concentrations. The stimulation of PTH secretion by high phosphate levels can be prevented by increases in intracellular calcium levels.

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