scholarly journals Redox modulation of intracellular free calcium concentration in thyroid FRTL-5 cells: evidence for an enhanced extrusion of calcium

1999 ◽  
Vol 339 (3) ◽  
pp. 621-628 ◽  
Author(s):  
Kid TÖRNQUIST ◽  
Petri VAINIO ◽  
Alexey TITIEVSKY ◽  
Benoit DUGUÉ ◽  
Raimo TUOMINEN
Keyword(s):  
Plasma Membrane ◽  
Calcium Concentration ◽  
Calcium Entry ◽  
Free Calcium ◽  
Redox Modulation ◽  
Kinase C ◽  
Store Operated Calcium Entry ◽  
Intracellular Free Calcium Concentration ◽  
Free Calcium Concentration ◽  
In Cells

Redox modulation is involved in the regulation of the intracellular free calcium concentration ([Ca2+]i) in several cell types. In thyroid cells, including thyroid FRTL-5 cells, changes in [Ca2+]i regulate important functions. In the present study we investigated the effects of the oxidizing compounds thimerosal and t-butyl hydroperoxide on [Ca2+]i in thyroid FRTL-5 cells. Thimerosal mobilized sequestered calcium, and evoked modest store-dependent calcium entry. Both compounds potently attenuated the increase in [Ca2+]i when store-operated calcium entry was evoked with thapsigargin. The entry of barium was not attenuated. Experiments performed with high extracellular pH, in sodium-free buffer and in the presence of vanadate suggested that thimerosal decreased [Ca2+]i by activating a calcium extrusion mechanism, probably a plasma membrane Ca2+-ATPase. All the observed effects were abrogated by the reducing agent β-mercaptoethanol. The mechanism of action was apparently mediated via activation of protein kinase C, as thimerosal potently stimulated binding of [3H]phorbol 12,13-dibutyrate, and was without effect on store-operated calcium entry in cells treated with staurosporine or in cells with down-regulated protein kinase C. Thimerosal did not depolarize the membrane potential, as evaluated using patch-clamp in the whole-cell mode. In immunoprecipitates obtained with an antibody against plasma membrane Ca2+-ATPase, we observed several phosphorylated bands in cells stimulated with thimerosal. In conclusion, we have shown that thimerosal attenuates an increase in [Ca2+]i, probably by activating a plasma membrane Ca2+-ATPase.

66 Orai1 IS REQUIRED TO MAINTAIN CALCIUM OSCILLATION AT FERTILIZATION IN PORCINE OOCYTES

2012 ◽  
Vol 24 (1) ◽  
pp. 145
Author(s):  
C. Wang ◽  
K. Lee
Keyword(s):  
Plasma Membrane ◽  
Embryo Development ◽  
Calcium Concentration ◽  
Calcium Influx ◽  
Calcium Entry ◽  
Intracellular Free Calcium ◽  
Calcium Oscillation ◽  
Free Calcium ◽  
Intracellular Free Calcium Concentration ◽  
Free Calcium Concentration

During mammalian fertilization, the sperm induces an oscillation in the oocyte's intracellular free calcium concentration that stimulates oocyte activation. The train of calcium spikes is maintained by an influx of calcium across the plasma membrane, probably through a mechanism known as store-operated calcium entry. Despite their importance, little is known about the identity and regulation of the calcium entry channels that mediate calcium influx at fertilization. Previously we have shown that the Orai1 protein is localised in the plasma membrane of pig oocytes and plays an important role in calcium entry after depletion of the intracellular calcium stores. In this study, we investigated the function of Orai1 in signal transduction during fertilization. In Experiment 1, Orai1 levels were down-regulated by injecting immature pig oocytes with small interfering RNA (siRNA) against Orai1; control cells were injected with scrambled siRNA. The injected oocytes were then matured in vitro for 44 h. In Experiment 2, Orai1 was overexpressed by microinjection of mRNA encoding Orai1 conjugated to the enhanced green fluorescent protein (EGFP-Orai1) 36 h after the beginning of maturation; these oocytes were incubated for an additional 8 h. At the end of the incubation period the oocytes were loaded with the calcium indicator dye fura-2 and inseminated. Changes in the intracellular free calcium concentration were monitored by means of a fluorescence imaging system. Some of the fertilized oocytes were cultured for 7 days, at which time embryo development together with the total nuclear number of the embryos were recorded. Data were subjected to 1-way ANOVA; differences between treatments were analysed using the Tukey test. The level of Orai1 protein was significantly lower in Orai1 siRNA-injected oocytes compared to the control group, as determined by Western blot, indicating successful down-regulation of Orai1. During fertilization, control oocytes displayed a series of calcium transients that lasted up to 8 h. However, in Orai1 siRNA-injected oocytes (9 out of 10 measured) only 1 calcium rise could be observed; these oocytes were unable to generate repetitive calcium signals after gamete fusion. Overexpression of Orai1 also disrupted the pattern of the normal fertilization calcium signal; the oscillation ceased after just a few Ca2+ rises in 8 out of 10 oocytes. The percentage of cleaved oocytes was lower in the siRNA-injected group compared to both non-injected or control siRNA-injected oocytes (38.4 ± 4.6% vs 70.5 ± 2.8% and 70.4 ± 2.4%; P < 0.05). The frequency of blastocyst (1.9 ± 0.8% vs11.0 ± 3.1% and 9.9 ± 1.4%) and the total cell number per blastocyst (19.8 ± 0.8 vs 36.2 ± 0.7 and 33.5 ± 5.5) was also significantly lower in oocytes with down-regulated Orai1 levels. These results indicate that an Orai1-mediated calcium influx is required to maintain proper calcium oscillation at fertilization and is also essential to support subsequent embryo development.

Role of Protein Kinase C in the Anti-Aggregatory Effects of Endothelin-1 on Human Platelets

1995 ◽  
Vol 88 (3) ◽  
pp. 277-283 ◽  
Author(s):  
R. M. Touyz ◽  
E. L. Schiffrin
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Calcium Concentration ◽  
Human Platelets ◽  
Intracellular Free Calcium ◽  
Free Calcium ◽  
Endothelin 1 ◽  
Kinase C ◽  
Intracellular Free Calcium Concentration ◽  
Free Calcium Concentration

1. Endothelin-1 has anti-aggregatory properties, but the mechanism underlying this inhibitory action is unknown. This in vitro study investigates effects of endothelin-1 on thrombin-stimulated aggregation and intracellular free calcium concentration in human platelets and assesses the role of protein kinase C in the interactions between endothelin-1 and thrombin. Aggregation was measured turbidometrically and the intracellular free calcium concentration was determined with the fluorescent indicator fura 2-acetoxymethyl ester. 2. Endothelin-1 at concentrations from 10−11 to 10−6 mol/l had no effect on platelet aggregation or intracellular free calcium concentration but inhibited in a dose-dependent manner aggregation induced by 0.05 unit/ml thrombin (pD2 for inhibition by endothelin = 8.1 ± 0.12). 3. Endothelin-1 at 10−9 mol/l significantly decreased (P<0.01) thrombin-stimulated aggregation from 81.4 ± 1.5% (in the absence of endothelin-1) to 53.5 ± 1.1% (in the presence of endothelin) and thrombin-stimulated intracellular free calcium concentration from 179 ± 1.7 nmol/l to 140 ± 1.8 nmol/l. 4. Preincubation of platelets with 10−7 mol/l staurosporine (protein kinase C inhibitor), calphostin C (highly selective protein kinase C inhibitor) or 5-(N,N-hexamethylene) amiloride (highly selective Na+-H+ exchange blocker) significantly inhibited (P < 0.01) thrombin-stimulated platelet responses and suppressed the inhibitory effect of endothelin-1 on thrombin-induced aggregation and intracellular free calcium concentration. 5. In conclusion, endothelin-1 decreases the aggregatory response of human platelets to thrombin by mechanisms that probably involve protein kinase C and Na+-H+ linked pathways.

Identification of ecto-PKC on surface of human platelets: role in maintenance of latent fibrinogen receptors

2000 ◽  
Vol 278 (6) ◽  
pp. H2008-H2019 ◽  
Author(s):  
Anna Babinska ◽  
Michael V. Hogan ◽  
Tomasz Sobocki ◽  
Malgorzata B. Sobocka ◽  
Yigal H. Ehrlich ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Calcium Concentration ◽  
Surface Protein ◽  
Human Platelets ◽  
Surface Proteins ◽  
Free Calcium ◽  
Platelet Surface ◽  
Inhibitory Peptides ◽  
Intracellular Free Calcium Concentration ◽  
Free Calcium Concentration

Human platelets express a protein phosphorylation system on their surface. A specific protein kinase C (PKC) antibody, monoclonal antibody (MAb) 1.9, which binds to the catalytic domain of PKC and inhibits its activity, causes the aggregation of intact platelets while inhibiting the phosphorylation of platelet surface proteins. Photoaffinity labeling with 100 nM 8-azido-[α32P]ATP identified this ecto-PKC as a single surface protein of 43 kDa sensitive to proteolysis by extracellular 0.0005% trypsin. Inhibition of the binding of 8-azido-[α32P]ATP to the 43-kDa surface protein by MAb 1.9 identified this site as the active domain of ecto-PKC. Covalent binding of the azido-ATP molecule to the 43-kDa surface protein inhibited the phosphorylative activity of the platelet ecto-PKC. Furthermore, PKC pseudosubstrate inhibitory peptides directly induced the aggregation of platelets and inhibited azido-ATP binding to the 43-kDa protein. Platelet aggregation induced by MAb 1.9 and by PKC inhibitory peptides required the presence of fibrinogen and resulted in an increase in the level of intracellular free calcium concentration. This increase in intracellular free calcium concentration induced by MAb 1.9 was found to be dependent on the binding of fibrinogen to activated GPIIb/IIIa integrins, suggesting that MAb 1.9 causes Ca2+flux through the fibrinogen receptor complex. We conclude that a decrease in the state of phosphorylation of platelet surface proteins caused by inhibition of ecto-PKC results in membrane rearrangements that can induce the activation of latent fibrinogen receptors, leading to platelet aggregation. Accordingly, the maintenance of a physiological steady state of phosphorylation of proteins on the platelet surface by ecto-PKC activity appears to be one of the homeostatic mechanisms that maintain fibrinogen receptors of circulating platelets in a latent state that cannot bind fibrinogen.

P2 purinoceptor localization along rat nephron and evidence suggesting existence of subtypes P2Y1 and P2Y2

1998 ◽  
Vol 274 (6) ◽  
pp. F1006-F1014 ◽  
Author(s):  
Seok Ho Cha ◽  
Takashi Sekine ◽  
Hitoshi Endou
Keyword(s):  
Calcium Concentration ◽  
Free Calcium ◽  
Dependent Manner ◽  
Reactive Blue 2 ◽  
Collecting Tubule ◽  
Single Nephron ◽  
Intracellular Free Calcium Concentration ◽  
Free Calcium Concentration ◽  
Dose Dependent ◽  
Cortical Collecting Tubule

Effects of extracellular ATP on intracellular free calcium concentration ([Ca2+]i) were examined in rat single nephron segments using the fura 2-AM. ATP (10 μM) induced a significant transient increase in [Ca2+]iin the glomerulus, the early proximal convoluted tubule (S1), the cortical collecting tubule (CCT), and the outer medullary collecting tubule (OMCT). The magnitude of the response was the greatest in the OMCT among four segments. ATP induced an increase in the [Ca2+]iin a dose-dependent manner in S1 and OMCT. In the OMCT, ATP caused a biphasic increase in [Ca2+]iconsisting of an initial rapid rise and a sustained phase. Removal of calcium from the medium resulted in an attenuation of the sustained phase of [Ca2+]iand an ∼30% reduction in the height of the initial [Ca2+]ipeak in response to 10 μM ATP. Effects of ATP, its analogs, and its metabolites were tested in the S1 and OMCT. ATP, 2-methylthio-ATP (2-MeS-ATP), ADP, and UTP increased [Ca2+]idose dependently. AMP and adenosine did not affect [Ca2+]iin the S1 and OMCT. The ATP- or 2-MeS-ATP-induced [Ca2+]iincrease was inhibited by the pretreatment of the S1 and OMCT with suramin or reactive blue 2. Neomycin, a phospholipase C inhibitor, attenuated the ATP-induced [Ca2+]iincrease. To investigate the hormonelike action of ATP in OMCT, a heterologous cross desensitization was performed. The pretreatment of OMCT with ATP inhibited increases in vasopressin-, ANG II-, endothelin-1-, or bradykinin-induced [Ca2+]iincrease. These findings suggest that ATP might affect the above peptidyl agonist-activated calcium mobilizations.

A cross-sectional study of basal platelet intracellular free calcium concentration in normotensive and hypertensive primigravid pregnancies

1990 ◽  
Vol 78 (1) ◽  
pp. 75-80 ◽  
Author(s):  
M. D. Kilby ◽  
F. Broughton Pipkin ◽  
S. Cockbill ◽  
S. Heptinstall ◽  
E. M. Symonds
Keyword(s):  
Calcium Concentration ◽  
Post Partum ◽  
Cross Sectional Study ◽  
Free Calcium ◽  
Sectional Study ◽  
Third Trimester ◽  
Cross Sectional ◽  
The Third ◽  
Intracellular Free Calcium Concentration ◽  
Free Calcium Concentration

1. The intracellular free calcium concentration ([Ca2+]i) in washed human platelets was measured using the fluorescent indicator, fura-2, in a cross-sectional study of 36 normotensive, primigravid volunteers, 12 in each trimester of pregnancy and a further 12 at 6 weeks post partum. The results were compared with those obtained from 30 normal female volunteers not using oral contraception. 2. The mean basal [Ca2+]i in the platelets of the pregnant women in the first two trimesters (115.6 ± 6.7 and 120.1 ± 5.7 nmol/l, respectively) was not shown to differ significantly from that of normal non-pregnant volunteers (112.3 ± 2.9 nmol/l). However, during the third trimester a significant increase in [Ca2+]i was noted (134.0 ± 4.9 nmol/l; P < 0.05), with a return to normal values in the post-partum period (108.2 ± 6.1 nmol/l). 3. [Ca2+]i was also measured in the platelets of a group of 12 primigravid pregnant women in the third trimester whose pregnancies were complicated by gestational hypertension (pregnancy-induced hypertension and preeclampsia). A significant rise in basal [Ca2+]i was noted in the platelets of primigravidae whose pregnancies were complicated by pre-eclampsia (163.6 ± 8.8 nmol/l) as compared with normotensive, third-trimester primigravidae (P < 0.02). However, no correlation could be demonstrated between [Ca2+]i and systemic blood pressure.

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