Mitogens induce calcium transients in both dividing and terminally differentiating keratinocytes

1991 ◽  
Vol 99 (2) ◽  
pp. 397-405
Author(s):  
F.M. Watt ◽  
D.L. Hudson ◽  
A.G. Lamb ◽  
S.R. Bolsover ◽  
R.A. Silver ◽  
...  
Keyword(s):  
Calcium Ions ◽  
Thymidine Incorporation ◽  
Calf Serum ◽  
Terminal Differentiation ◽  
Free Calcium ◽  
Imaging Method ◽  
Standard Medium ◽  
Low Calcium ◽  
Calcium Indicator ◽  
Differentiation Status

During terminal differentiation, keratinocytes lose the ability to divide. One indicator of responsiveness to certain growth factors is a transient rise in the intracellular concentration of free calcium ions ([Ca2+]i). The aim of our experiments was to discover whether or not terminally differentiating keratinocytes have lost the ability to exhibit an increase in [Ca2+]i in response to factors that stimulate [3H]thymidine incorporation and increase [Ca2+]i in undifferentiated keratinocytes. [Ca2+]i was measured with the calcium indicator dye FURA-2 and by a ratio imaging method. Expression of involucrin, a precursor of the keratinocyte cornified envelope, was used as a marker of terminal differentiation. Measurements were made on stratified colonies of cells grown in standard medium (containing 1.8 mM calcium ions) and on cell monolayers in low calcium medium (0.1 mM). Treatment of serum-starved monolayers with substance P, bombesin or complete growth medium containing 10% fetal calf serum resulted in increased [3H]thymidine incorporation. A switch from low calcium to standard medium also stimulated [3H]thymidine incorporation whether or not the cells had been serum-starved. In each experiment some cells showed an increase in [Ca2+]i while others did not. However, the heterogeneity in the [Ca2+]i response did not reflect the terminal differentiation status of individual cells: both involucrin-positive and -negative cells were found in the responding and nonresponding populations. Involucrin-positive and -negative areas of stratified cultures also underwent a transient increase in [Ca2+]i in response to serum-containing medium. Our data therefore indicate that both proliferating (involucrin-negative) and post-mitotic, terminally differentiating (involucrin-positive) keratinocytes can respond to mitogenic stimuli by an increase in [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Evidence that cadherins play a role in the downregulation of integrin expression that occurs during keratinocyte terminal differentiation

1994 ◽  
Vol 124 (4) ◽  
pp. 589-600 ◽  
Author(s):  
KJ Hodivala ◽  
FM Watt
Keyword(s):  
De Novo ◽  
Lectin Binding ◽  
Basal Layer ◽  
Terminal Differentiation ◽  
Ion Concentration ◽  
Epidermal Keratinocytes ◽  
Integrin Expression ◽  
Standard Medium ◽  
Proliferating Cells ◽  
Low Calcium

In epidermis the onset of terminal differentiation normally coincides with inhibition of integrin function and expression, thereby ensuring that differentiating cells are selectively expelled from the basal layer. However, when stratification of cultured human epidermal keratinocytes is prevented by reducing the calcium concentration of the medium to 0.1 mM, keratinocytes initiate terminal differentiation while still attached to the culture substrate. We have examined the mechanism by which differentiating keratinocytes adhere to extracellular matrix proteins in low calcium medium and the consequences of inducing stratification by raising the calcium ion concentration to 1.8 mM (Standard Medium). In low calcium medium keratinocytes co-expressed integrins and terminal differentiation markers such as involucrin and peanut lectin-binding glycoproteins: differentiating cells contained integrin mRNA, synthesized integrin proteins de novo and expressed functional mature integrins. There were no differences in integrin synthesis, maturation or break down in low calcium or standard medium, although the level of beta 1 integrins on the surface of proliferating cells was higher in standard medium. Within 6 h of transfer from low calcium to standard medium integrin mRNA was no longer detectable in terminally differentiating cells, integrins were being lost from the cell surface, and selective migration out of the basal layer had begun. Antibodies to P- and E-cadherin, which block calcium-induced stratification, prevented the selective loss of integrin mRNA and protein from terminally differentiating cells. This suggests that cadherins may play a role in the down-regulation of integrin expression that is associated with terminal differentiation.

Mitochondrial matrix calcium ions regulate energy production in myocardium: A cytochemical study

1990 ◽  
Vol 48 (2) ◽  
pp. 166-167
Author(s):  
W.A. Jacob ◽  
R. Hertsens ◽  
A. Van Bogaert ◽  
M. De Smet
Keyword(s):  
Energy Metabolism ◽  
Calcium Ions ◽  
Energy Production ◽  
Regulation Of Respiration ◽  
Free Calcium ◽  
Mitochondrial Matrix ◽  
Cytochemical Study ◽  
Nmr Techniques

In the past most studies of the control of energy metabolism focus on the role of the phosphorylation potential ATP/ADP.Pi on the regulation of respiration. Studies using NMR techniques have demonstrated that the concentrations of these compounds for oxidation phosphorylation do not change appreciably throughout the cardiac cycle and during increases in cardiac work. Hence regulation of energy production by calcium ions, present in the mitochondrial matrix, has been the object of a number of recent studies.Three exclusively intramitochondnal dehydrogenases are key enzymes for the regulation of oxidative metabolism. They are activated by calcium ions in the low micromolar range. Since, however, earlier estimates of the intramitochondnal calcium, based on equilibrium thermodynamic considerations, were in the millimolar range, a physiological correlation was not evident. The introduction of calcium-sensitive probes fura-2 and indo-1 made monitoring of free calcium during changing energy metabolism possible. These studies were performed on isolated mitochondria and extrapolation to the in vivo situation is more or less speculative.

scholarly journals Intracellular free calcium oscillates during cell division of Xenopus embryos.

1991 ◽  
Vol 112 (4) ◽  
pp. 711-718 ◽  
Author(s):  
N Grandin ◽  
M Charbonneau
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Cell Volume ◽  
Intracellular Ph ◽  
Calcium Ions ◽  
Volume Ratio ◽  
Mean Amplitude ◽  
Free Calcium ◽  
Periodic Oscillations ◽  
Xenopus Embryos

In Xenopus embryos, previous results failed to detect changes in the activity of free calcium ions (Ca2+i) during cell division using Ca2(+)-selective microelectrodes, while experiments with aequorin yielded uncertain results complicated by the variation during cell division of the aequorin concentration to cell volume ratio. We now report, using Ca2(+)-selective microelectrodes, that cell division in Xenopus embryos is accompanied by periodic oscillations of the Ca2+i level, which occur with a periodicity of 30 min, equal to that of the cell cycle. These Ca2+i oscillations were detected in 24 out of 35 experiments, and had a mean amplitude of 70 nM, around a basal Ca2+i level of 0.40 microM. Ca2+i oscillations did not take place in the absence of cell division, either in artificially activated eggs or in cleavage-blocked embryos. Therefore, Ca2+i oscillations do not represent, unlike intracellular pH oscillations (Grandin, N., and M. Charbonneau. J. Cell Biol. 111:523-532. 1990), a component of the basic cell cycle ("cytoplasmic clock" or "master oscillator"), but appear to be more likely related to some events of mitosis.

scholarly journals Cytosolic free calcium changes induced by chemotactic peptide in neutrophils from patients with chronic granulomatous disease

Blood ◽  
1984 ◽  
Vol 63 (1) ◽  
pp. 231-233 ◽  
Author(s):  
PD Lew ◽  
C Wollheim ◽  
RA Seger ◽  
T Pozzan
Keyword(s):  
Chronic Granulomatous Disease ◽  
Calcium Concentration ◽  
Cytosolic Calcium ◽  
Human Neutrophils ◽  
Free Calcium ◽  
Granulomatous Disease ◽  
Chemotactic Peptide ◽  
Intact Cells ◽  
Calcium Indicator ◽  
Free Calcium Concentration

Abstract Cytoplasmic free calcium concentration (Ca2+)i was measured in neutrophils from patients with the classical X-linked form of chronic granulomatous disease (CGD) by trapping the fluorescent calcium indicator Quin 2 in intact cells. CGD neutrophils do not produce superoxide and are only slightly depolarized upon stimulation by the chemotactic peptide. N-formyl-methionyl-leucyl-phenylalanine (FMLP). The resting levels, as well as (Ca2+)i changes induced by FMLP in CGD cells, were quantitatively and kinetically similar to those observed in normal cells. We conclude that the defect in CGD cells is distal to, or independent of, the changes in (Ca2+)i induced by FMLP stimulation and that normal membrane depolarization does not seem to be necessary for receptor-mediated rise in free cytosolic calcium in human neutrophils.

Estrogen reduces proliferation and agonist-induced calcium increase in coronary artery smooth muscle cells

1997 ◽  
Vol 272 (4) ◽  
pp. H1996-H2003 ◽  
Author(s):  
R. C. Bhalla ◽  
K. F. Toth ◽  
R. A. Bhatty ◽  
L. P. Thompson ◽  
R. V. Sharma
Keyword(s):  
Cell Proliferation ◽  
Smooth Muscle ◽  
Coronary Artery ◽  
Guinea Pig ◽  
Untreated Control ◽  
Fetal Calf Serum ◽  
Thymidine Incorporation ◽  
Calf Serum ◽  
Causative Role ◽  
Pig Coronary Artery

Epidemiological evidence and estrogen replacement studies suggest that estrogen has a protective effect on the cardiovascular system against coronary artery disease. Vascular smooth muscle (VSM) cell replication has been shown to play a causative role in the pathogenesis of atherosclerosis. Therefore, in this study, we investigated the effect of chronic treatment of cultured guinea pig coronary artery VSM cells with physiological concentrations of 17beta-estradiol (E2) on thymidine incorporation, cell proliferation, and bradykinin-stimulated cytosolic calcium concentration ([Ca2+]i). Bradykinin at physiological concentrations causes contraction of endothelium-denuded guinea pig coronary artery rings in a concentration-dependent manner. VSM cells were first treated with low doses of E2 (10 pg/ml) for 1-2 days followed by treatment for 4-6 days with 50 pg/ml of E2, a concentration similar to that found in pregnancy. Using these protocols, we consistently observed the presence of E2-receptor mRNA in VSM cells by a ribonuclease protection assay. Fetal calf serum-stimulated [3H]thymidine incorporation was significantly reduced (P < 0.05) in E2-treated cells compared with untreated control cells. Similarly, E2 treatment significantly inhibited fetal calf serum-stimulated VSM cell proliferation compared with untreated control cells (P < 0.05). We also tested the hypothesis that E2 treatment attenuates agonist-stimulated [Ca2+]i in VSM cells because acute E2 treatment has been shown to produce relaxation of precontracted isolated coronary artery preparations. E2 treatment of VSM cells resulted in a significant decrease in bradykinin-stimulated [Ca2+]i compared with untreated cells (P < 0.05). In conclusion, our data demonstrate that estrogen at physiological concentrations directly regulates coronary VSM cell function.

Differential Involvement of Ca2+ Channels in Survival and Neurite Outgrowth of Cultured Embryonic Cockroach Brain Neurons

2002 ◽  
Vol 88 (3) ◽  
pp. 1475-1490 ◽  
Author(s):  
Pascal Benquet ◽  
Janine Le Guen ◽  
Yves Pichon ◽  
François Tiaho
Keyword(s):  
Cell Death ◽  
Calcium Channels ◽  
Neurite Outgrowth ◽  
Calcium Current ◽  
Culture Media ◽  
Calf Serum ◽  
Free Calcium ◽  
Extracellular Potassium ◽  
Patch Clamp Technique ◽  
Brain Neurons

The contribution of voltage-gated calcium channels (VGCC) to the development of cultured embryonic cockroach brain neurons was assessed using pharmacological agents. VGCC currents were recorded using the patch-clamp technique and were found to be blocked dose-dependently by micromolar concentrations of mibefradil. The activation and inactivation properties of the calcium channels enable a sizeable calcium current to flow at rest (about −30 and −20 mV in high-potassium culture media). As expected, the cytoplasmic-free calcium concentration was found to rise when the extracellular potassium concentration was raised from 3 to 15 and 30 mM. The effects of VGCC blockers and calcium chelators were different in fresh and in mature cultures in which the neurons were connected to each other to form a defined network. In fresh cultures, the two non-selective VGCC blockers (verapamil and mibefradil) induced a dose-dependent cell death that was proportional to their blocking effect on I Ba. This effect could not be prevented by addition of fetal calf serum to the culture medium. A similar effect was obtained using intra- or extracellular calcium chelating agents (10 μM BAPTA-AM or 10 mM EGTA). Quite unexpectedly, blockade of the P/Q-like (ω-Aga WA-sensitive) component of the calcium current by 500 nM of ω-AgaTx IVA had no lethal effect, suggesting that the corresponding channels are not involved in the survival mechanism. As expected from their lack of effect on I Ba, isradipine, nifedipine, and ω-CgTx GVIA did not induce cell death. When the neurons started growing neurites, their sensitivity to calcium channel blockade by mibefradil decreased, indicating a correlation between neurite outgrowth and resistance to calcium depletion. In mature cultures, the neurons became resistant to mibefradil, verapamil, and BAPTA-AM. However, these agents, as well as ω-AgaTx IVA, had a significant inhibitory effect on the increase in diameter of the connectives that linked adjacent clusters of neurons. This effect has been shown to result, in the case of mibefradil, from an inhibition of neurite outgrowth characterized by a significant reduction of the number of primary neurites and secondary branchings but not to a significant modification of the diameter of individual neurites. These results support the view that, as in vertebrates, calcium influx through VGCC plays an important role in survival and neurite outgrowth of cultured embryonic insect neurons. The differential contribution of the P/Q-like and R-like (ω-Aga WA-sensitive) calcium channels in these processes is discussed.

Cytosolic free Calcium and Intracellular Calcium Stores in Neutrophils from Hypertensive Subjects

1985 ◽  
Vol 69 (2) ◽  
pp. 227-230 ◽  
Author(s):  
P. Daniel Lew ◽  
Laurent Favre ◽  
Francis A. Waldvogel ◽  
Michel B. Vallotton
Keyword(s):  
Essential Hypertension ◽  
Intracellular Calcium ◽  
Calcium Ionophore ◽  
Calcium Handling ◽  
Free Calcium ◽  
Calcium Stores ◽  
Cytosolic Free Calcium ◽  
Intact Cells ◽  
Intracellular Calcium Stores ◽  
Calcium Indicator

1. Alterations in intracellular calcium have been implicated in the pathogenesis of essential hypertension. To see whether this is a generalized phenomenon we assessed cytosolic free calcium and intracellular calcium stores in neutrophils from normo- and hyper-tensive subjects, by trapping the fluorescent calcium indicator quin2 in intact cells. 2. Ten patients with untreated essential hypertension were compared with 10 age- and sex-matched normotensive subjects. The levels of cytosolic free calcium and intracellular calcium stores releasable by the calcium ionophore ionomycin did not differ. No significant relationship was found between blood pressure and the calcium parameters in all 20 subjects studied. 3. The results indicate that essential hypertension is not associated with a membrane defect in calcium handling of all human cell systems, leading to generalized increases in resting values of cytosolic free calcium. 4. Neutrophils do not appear to be a good model for intracellular calcium handling in vascular smooth muscle.

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