Calcium ion imaging in plant cells

1993 ◽  
Vol 51 ◽  
pp. 132-133
Author(s):  
Peter K. Hepler ◽  
Dale A. Callaham
Keyword(s):  
Plant Cell Wall ◽  
Cytoplasmic Membrane ◽  
Fluorescent Dyes ◽  
Free Acid ◽  
Methyl Esters ◽  
Free Calcium ◽  
Local Concentration ◽  
Ion Imaging ◽  
Membrane Compartments ◽  
Free Acid Form

Calcium ions (Ca) participate in many signal transduction processes, and for that reason it is important to determine where these ions are located within the living cell, and when and to what extent they change their local concentration. Of the different Ca-specific indicators, the fluorescent dyes, developed by Grynkiewicz et al. (1), have proved most efficacious, however, their use on plants has met with several problems (2). First, the dyes as acetoxy-methyl esters are often cleaved by extracellular esterases in the plant cell wall, and thus they do not enter the cell. Second, if the dye crosses the plasma membrane it may continue into non-cytoplasmic membrane compartments. Third, even if cleaved by esterases in the cytoplasm, or introduced as the free acid into the cytoplasmic compartment, the dyes often become quickly sequestered into vacuoles and organelles, or extruded from the cell. Finally, the free acid form of the dye readily complexes with proteins reducing its ability to detect free calcium. All these problems lead to an erroneous measurement of calcium (2).

Factors Influencing the Measurement of Calcium Concentrations in Biological Fluids by 19F N.M.R. Spectroscopy

1994 ◽  
Vol 47 (4) ◽  
pp. 677
Author(s):  
GL Mendz
Keyword(s):  
Chemical Exchange ◽  
Free Acid ◽  
Biological Fluids ◽  
Model Systems ◽  
Free Calcium ◽  
Acid Form ◽  
Tetraacetic Acid ◽  
Factors Influencing ◽  
Free Acid Form ◽  
Derivatives Of

Fluorinated derivatives of fluorescent calcium probes have been recently used to measure free calcium concentrations in biological fluids by 19F n.m.r. spectroscopy. To measure calcium levels a method utilizes the change in chemical shift experienced by the fluorine resonance of the calcium chelator 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′- tetraacetic acid upon complexation of cations. The procedure assumes that (A) the fluorinated probe is in equilibrium between the calcium complex and the free acid form; (B) the chemical exchange between different sites has similar effects on the intensities of the observable resonances; and (c) the presence of macromolecules affects equally the intensities of the resonances arising from different complexes. Experiments designed to test these assumptions were carried out in plasma and in model systems containing mixtures of cations and macromolecules. The results indicated that in biological fluids the calcium probe is involved in a multisite equilibrium in which 'invisible' (very broad) peaks affect the equilibrium intensities of the 'observable' peaks. Binding to macromolecules affected differently the intensities of the resonances arising from different complexes in the spectrum of the chelator , and influenced significantly the calculated free calcium concentrations.

An ATP-sensitive conductance in cultured smooth muscle cells from pregnant rat myometrium

1989 ◽  
Vol 257 (2) ◽  
pp. C297-C305 ◽  
Author(s):  
E. Honore ◽  
C. Martin ◽  
C. Mironneau ◽  
J. Mironneau
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Voltage Clamp ◽  
Free Acid ◽  
Muscle Cells ◽  
Monovalent Cation ◽  
Acid Form ◽  
Pregnant Rat ◽  
Free Acid Form ◽  
Cultured Smooth Muscle Cells

The whole cell voltage-clamp technique was used to study the effects of extracellular ATP in cultured smooth muscle cells isolated from pregnant rat myometrium. An inward current was elicited by ATP (IATP) in cells held at -70 mV under voltage clamp. The amplitude of IATP was reduced by estrogen pretreatment and by the end of pregnancy. IATP not only did not undergo any desensitization but showed facilitation. The current-voltage relationship of IATP was linear and reversed close to 0 mV. Changing the sodium electrochemical gradient by decreasing extracellular or intracellular sodium resulted in a linear relationship between the reversal potential of IATP and Na equilibrium potential that, however, differed from the predicted curve for a purely sodium conductance. The conductance activated by ATP was monovalent cation selective with little discrimination between potassium, cesium, and sodium ions. IATP was depressed by divalent cations, and the rank order of potency was Co greater than Mg greater than Ca greater than Ba, suggesting that the free-acid form of ATP was the effective ligand. Adenosine, AMP, and ADP were ineffective in eliciting IATP, whereas ATP gamma S and alpha,beta-methylene ATP were capable of mimicking the effects of ATP, although they were less potent. These results are consistent with the free-acid form of ATP activating a monovalent cation-selective and estrogen-sensitive conductance in myometrium.

scholarly journals Intracellular calcium mobilization and activation of the Na+/H+ exchanger in platelets

1993 ◽  
Vol 290 (2) ◽  
pp. 617-622 ◽  
Author(s):  
E Poch ◽  
A Botey ◽  
J Gaya ◽  
A Cases ◽  
F Rivera ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Fluorescent Dyes ◽  
Extracellular Calcium ◽  
Free Calcium ◽  
Regulatory Relationship ◽  
Cytosolic Ph ◽  
Intracellular Calcium Mobilization ◽  
Free Calcium Concentration ◽  
Cytosolic Free Calcium Concentration ◽  
Intracellular Alkalinization

The aim of the present study was to evaluate the regulatory relationship between the cytosolic free calcium concentration ([Ca2+]i and cytosolic pH (pHi). [Ca2+]i and pHi were measured using the fluorescent dyes fura-2 and BCECF [2′,7′-bis-(carboxyethyl)-5,6-carboxyfluorescein] respectively. In a medium with 1 mmol/l extracellular calcium, thrombin (2.5 units/ml) induced an increment in [Ca2+]i of 638 +/- 31 nmol/l (n = 5) and an intracellular alkalinization of 0.14 +/- 0.01 pH units (n = 8). Both responses were dependent on the concentration of thrombin, displaying a sigmoidal dose-response pattern. The intracellular alkalinization was dependent upon extracellular Na+ and was amiloride-sensitive, indicating that it was mediated by activation of the Na+/H+ exchanger. When extracellular calcium was chelated with EGTA prior to the addition of thrombin, the intracellular alkalinization was not affected (0.15 +/- 0.02 at 2.5 units/ml thrombin, n = 8). Under these circumstances, the [Ca2+]i increment represents mobilization from internal stores, reaching 157 +/- 42 nmol/l at 2.5 units/ml thrombin. When platelets were preloaded with the intracellular calcium chelator MAPTAM (1,2-bis-5-methylaminophenoxylethane-NNN'-tetraacetoxymethyl acetate) to block the increase in [Ca2+]i induced by thrombin, no increment in pHi was observed. Moreover, MAPTAM-loaded calcium-depleted platelets had a basal pHi that was more acidic than in the presence of 1 mmol/l extracellular calcium (6.93 +/- 0.09 versus 7.14 +/- 0.01, n = 26, P < 0.001). Ionomycin induced an elevation of [Ca2+]i that was accompanied by a concomitant increase in pHi, which was Na(+)-dependent and amiloride-sensitive. [Ca2+]i and pHi increases induced by ionomycin were both dependent on the concentration of ionomycin. In conclusion, an increase in [Ca2+]i is necessary for the agonist-induced activation of the Na+/H+ exchanger in platelets. Non-agonist-induced increases in [Ca2+]i seems to prompt activation of the exchanger. In addition, Ca(2+)-depleted platelets have a more acidic basal pHi, indicating that the basal level of [Ca2+]i is also important for maintaining the basal pHi.

scholarly journals Microinjection of Ca++-calmodulin causes a localized depolymerization of microtubules.

1983 ◽  
Vol 97 (6) ◽  
pp. 1918-1924 ◽  
Author(s):  
C Keith ◽  
M DiPaola ◽  
F R Maxfield ◽  
M L Shelanski
Keyword(s):  
Mitotic Spindle ◽  
Calcium Ion ◽  
Molar Ratio ◽  
Free Calcium ◽  
Local Concentration ◽  
Dependent Manner ◽  
Calcium Dependent ◽  
Molar Ratios ◽  
Microtubule Polymerization ◽  
Full Complement

The microinjection of calcium-saturated calmodulin into living fibroblasts causes the rapid disruption of microtubules and stress fibers in a sharply delimited region concentric with the injection site. This effect is specific to the calcium-bearing form of calmodulin; neither calcium-free calmodulin nor calcium ion at similar levels affects the cytoskeleton. If cells have previously been microinjected with calcium-free calmodulin, elevation of their intracellular calcium levels to 25 mM potentiates the disruption of microtubules throughout the cytoplasm. Approximately 400 mM free calcium is required to cause an equivalent disruption in uninjected cells. The level of calmodulin necessary to disrupt the full complement of cellular microtubules is found to be approximately in 2:1 molar ratio to tubulin dimer. These results indicate that calmodulin can be localized within the cytoplasm in a calcium-dependent manner and that it can act to regulate the calcium lability of microtubules at molar ratios that could be achieved locally within the cell. Our results are consistent with the hypothesis that calmodulin may be controlling microtubule polymerization equilibria in areas of high local concentration such as the mitotic spindle.

EFFECTS OF VARIOUS CHEMICAL AND COLD-HARDENING TREATMENTS ON RESISTANCE OF SUGAR BEET SEEDLINGS TO FREEZING

1956 ◽  
Vol 34 (1) ◽  
pp. 154-158
Author(s):  
William G. Corns
Keyword(s):  
Sugar Beet ◽  
Sodium Salt ◽  
Free Acid ◽  
Cold Hardening ◽  
Chemically Treated ◽  
Free Acid Form ◽  
Series Of Experiments ◽  
The Difference ◽  
And Control ◽  
Low Temperature Resistance

Either the free acid form or the sodium salt of Dalapon (2,2-dichloropropionic acid) and of TCA (trichloroacetic acid) and the sodium salt of 2,2,3-trichloropropionic acid (free acid not tested) were effective in improving the low temperature resistance of sugar beet seedlings grown in 4- and 8-p.p.m. solutions in the dark at 21 °C., and evaluated by short exposures to −10 °C. Isopropy-N(3-chlorophenyl) carbamate, amino triazole, sodium chloride, and trichlorobenzoic acid were ineffective in similar tests. In a series of experiments involving periodic sampling and freezing of Dalapon-treated illuminated sugar beet seedlings during a 24 day period of storage at 6 °C., the chemically treated plants were again superior to the comparable controls. The "cold-hardening" treatments tended to increase the magnitude of the difference between chemically treated and control plants. The amount of improvement was more variable in the tests with green plants than with those grown in the dark.

Intracellular ion imaging using fluorescent dyes: artefacts and limits to resolution

1992 ◽  
Vol 420 (5-6) ◽  
pp. 595-602 ◽  
Author(s):  
R. Angus Silver ◽  
Michael Whitaker ◽  
Stephen R. Bolsover
Keyword(s):  
Fluorescent Dyes ◽  
Ion Imaging

ChemInform Abstract: Novel Solid-State 8H+-Heteropolyacid. Synthesis and Molecular Structure of a Free-Acid Form of a Dawson-Type Sandwich Complex, [Ti2{P2W15O54 (OH2)2}2]8-.

ChemInform ◽  
2008 ◽  
Vol 39 (7) ◽  
Author(s):  
Hideyuki Murakami ◽  
Kunihiko Hayashi ◽  
Ikuo Tsukada ◽  
Takeshi Hasegawa ◽  
Shoko Yoshida ◽  
...  
Keyword(s):  
Molecular Structure ◽  
Solid State ◽  
Free Acid ◽  
Acid Form ◽  
Sandwich Complex ◽  
Free Acid Form

scholarly journals Studies on the Mode of Action of Reutericyclin

2003 ◽  
Vol 69 (2) ◽  
pp. 1305-1307 ◽  
Author(s):  
Michael G. Gänzle ◽  
Rudi F. Vogel
Keyword(s):  
Mode Of Action ◽  
Cytoplasmic Membrane ◽  
Fluorescent Dyes ◽  
Large Molecules ◽  
Proton Potential

ABSTRACT The mode of action of reutericyclin was determined with fluorescent dyes that probed the permeability of the cytoplasmic membrane by large molecules, protons, and potassium. A comparison of reutericyclin activity with those of nisin, nigericin, and valinomycin demonstrated that reutericyclin does not form pores but selectively dissipates the transmembrane proton potential.

Ratio confocal imaging of free cytoplasmic calcium gradients in polarising and polarisedFucuszygotes

Zygote ◽  
1993 ◽  
Vol 1 (1) ◽  
pp. 9-15 ◽  
Author(s):  
Frederic Berger ◽  
Colin Brownlee
Keyword(s):  
Cell Division ◽  
Brown Alga ◽  
Free Acid ◽  
Confocal Imaging ◽  
Acid Form ◽  
Cytoplasmic Calcium ◽  
Molecular Weights ◽  
Free Acid Form ◽  
Marine Brown Alga ◽  
Calcium Green

SummaryIn the marine brown alga,Fucus, two poles are differentiated before cell division determining the future rhizoid or thallus. We have used a combination of the Ca2+-sensitive dye Calcium Green and the pH-sensitive dye SNARF monitored at pH-insensitive wavelengths to obtain confocal ratio images of free cytoplasmic calcium distribution at different stages in polarisingFucuszygotes. These dyes have the advantage that they can be used in most confocal microscopes and their longer excitation wavelengths greatly reduce autofluorescence problems. Dyes of varying molecular weights (free acid form, 10 000 mol.wt or 70 000 mol.wt dextran-conjugated) were pressure microinjected into early zygotes which were allowed to polarise in unidirectional light. Dextran-conjugated dyes remained non-compartmentalised and fluorescence could be monitored for up to 3 days following microinjection. Currently we have been able to detect Ca2+gradients at the tip of the rhizoid, confirming earlier results. Localised Ca2+elevations have also been observed at the rhizoid pole of the polarising zygote before the onset of rhizoid germination. Limitations of this technique and the significance of these Ca2+gradients are discussed.

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