The effects of pH and temperature on fluorescent calcium indicators as determined with Chelex-100 and EDTA buffer systems

Lattanzio, Frank A.

doi:10.1016/0006-291x(90)91362-v

Cited by 142 publications

(68 citation statements)

References 12 publications

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“…Our unpublished ratio measurements of BCECF in growing Agapanthus pollen tubes have shown that the pH is lower in the tip than behind it. Low pH decreases the dissociation constant of the Ca2+-dyes (Lattanzio, 1990) and increased protonation causes greater quenching of dyes such as CG-1 which are based on fluorescein chromophores (Minta et a/., 1989). Fluorescence ratio imaging can overcome many of these problems because the ratio is independent of such factors as unequal dye concentration, variations in the amount of cytosol, and variable dye quenching (Read eta/., 1992).…”

Section: Discussionmentioning

confidence: 99%

Role of cytosolic free calcium in the reorientation of pollen tube growth

et al. 1994

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Summary Growing pollen tubes of Agapanthus umbellatus exhibited a tip‐to‐base gradient in cytosolic free calcium ([Ca2+]c). Although this gradient is believed to be involved in pollen tube growth, its role in specifying reorientation is unknown. The direction of pollen tube growth could be modified by iontophoretic micro‐injection, electrical fields (EFs) or photolysis of caged Ca2+. Iontophoretic injection resulted in a temporary cessation of growth, an increase in [Ca2+]c and, upon recovery, reoriented growth. Weak EFs also elevated [Ca2+]c, reduced growth rates and resulted in the reorientation of pollen tubes towards the cathode. Treatment with very low concentrations of the Ca2+‐channel blocker lanthanum chloride, prior to exposure to an EF, inhibited both the increase in [Ca2+]c and reorientation whilst only slightly affecting growth rates. The responses of growth inhibition and reorientation were mimicked when [Ca2+]c was artificially elevated by photoactivating caged Ca2+ (Nitr‐5). Our data suggest that [Ca2+]c is part of a transduction mechanism which enables growing pollen tubes to successfully reorient to directional signals in the style and thus accomplish eventual fertilization of the egg.

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Section: Discussionmentioning

confidence: 99%

Role of cytosolic free calcium in the reorientation of pollen tube growth

et al. 1994

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“…Ratiometric analysis and display were performed off-line, using data only from voxels in which the intensity levels detected in both channels were more than twice background. Ratio values are displayed in pseudocolour using an optimized spectrum (Clarke & Leonard, 1989 Lattanzio, 1990) in the calibration equation (Grynkiewicz et al 1985) showing a high degree of reliability provided optical alignments and evenness of field illumination were optimized. In 8itu calibrations of indo-1 ratio measurements from minimum Ca2+ loading to saturation gave an effective range of ratios from 0f01 to 2f5, respectively (n = 7).…”

Section: Methodsmentioning

confidence: 99%

“…The apparent Kd of indo-1 for Ca2+ is pH sensitive (Lattanzio, 1990). Changes in pH were measured in control experiments using SNAFL-calcein AM.…”

Section: Methodsmentioning

confidence: 99%

“…The ratio value for each voxel (a temporal and spatial mean) can be transformed into an estimated value of Ca2+ activity given a knowledge of the maximal and minimal signals and the apparent Kd of indo-1 in the intracellular environment. Using the apparent Kd value of 236 nM (Lattanzio, 1990; pH 7 0) and the equations of Grynkiewicz et al (1985), the range of Ca2+ activities would be from < 100 nm under control conditions in most cells to > 500 nm in the presence of 1S,3R-ACPD. The colour bar is illustrated in the inset to The responses were shown to be concentration dependent by a series of experiments in which neurones were exposed to 0 3, 1, 30, 100 or 1000 ,M IS,3R-ACPD.…”

Section: S 3b-acpd Causes Elevation In Acai In Hippocampal Neuronesmentioning

confidence: 99%

“…The fluorescence ratios of the indo-1 within the neurones were found to be uniform and proportional to the level of [Ca2+]., allowing the calculation of [Ca2+]i (Grynkiewicz et al 1985). However, an important caveat is that the apparent Kd of indo-1 for Ca2+ is pH sensitive (Lattanzio, 1990). Thus changes in fluorescence ratio may reflect pH as well as aCai changes (Morris, Weigmann, Welling & Chronwall, 1994).…”

Section: /Sm 1s3r-acpd (Open Bars) the Individual Cells Havementioning

confidence: 99%

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Increased perinuclear Ca2+ activity evoked by metabotropic glutamate receptor activation in rat hippocampal neurones.

Phenna

Jane

Chad

1995

The Journal of Physiology

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1. The effect of metabotropic glutamate receptor activation on intracellular Ca2+ activity (alpha Cai) of rat hippocampal pyramidal neurones in vitro was examined using ratiometric confocal laser scanning microscopy with the Ca(2+)‐sensitive fluorescent probe indo‐1 AM. 2. Metabotropic receptors were selectively activated with 1S,3R‐1‐aminocyclopentane‐1,3‐dicarboxylic acid (1S,3R‐ACPD; 100 microM) in the presence of D‐2‐amino 5‐phosphonovaleric acid (D‐APV), 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) and CdCl2. Most pyramidal neurones (77/84) responded with an elevation in Ca2+ activity, maximal after 3‐5 min. Fluorescence ratio responses were concentration dependent (EC50 approximately 10 microM) and were blocked by prior application of the antagonist (RS)‐4‐carboxy‐3‐hydroxyphenylglycine (RS‐CHPG, 300 microM). 3. Responses to 1S,3R‐ACPD (100 microM) also caused acidification of the neurones, from estimated control pH 7.2 to pH 6.6 (measured with the pH‐sensitive dye SNAFL‐calcein). The correction factor for indo‐1 determination of Ca2+ was estimated to be x 1.4. 4. Elevations in alpha Cai were greater within the perinuclear region (> 1000 nM), than in the cytoplasm (approximately 200 nM). This region was devoid of staining by the endoplasmic reticulum staining dye 3,3'‐dihexyloxacarbocyanine iodide (DiOC6(3)). 5. It is concluded that activation of metabotropic receptors in immature rat hippocampal pyramidal neurones leads to a large increase in perinuclear Ca2+ which would be well positioned to interact with the genome.

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Calcium-independent effects of cadmium on actin assembly in mesangial and vascular smooth muscle cells

Wang

Chin

Templeton

1996

Cell Motil. Cytoskeleton

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Several metal ions are known to cause depolymerization of the actin cytoskeleton under some circumstances. We found that in renal mesangial and vascular smooth muscle cells, micromolar concentrations of Cd2+ result in loss of phalloidin‐stainable filamentous (F‐) actin. The decrease in F‐actin was not accompanied by a corresponding increase in G‐actin. The decrease in total actin could be accounted for in part by an inhibition by Cd2+ of total protein (and actin) synthesis after 6 to 8 h without an effect on actin degradation, and the equilibrium between F‐ and G‐actin was shifted to maintain near‐constant levels of G‐actin. However, Cd2+ caused significant decreases in F‐actin at earlier times, indicating effects on the polymerization equilibrium independent of those on actin synthesis. Only picomolar concentrations of free intracellular Cd2+ occur in these experiments. However, it is this Cd2+ pool which is responsible for F‐actin depolymerization because equal cellular concentrations of cadmium delivered as Cd‐metallothionein have no effect. The effect is also very specific for Cd2+ and under the same conditions neither Mg2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, nor Hg2+ result in any loss of F‐actin. Addition of Cd2+ to mesangial and vascular smooth muscle cells had no immediate effect on free intracellular calcium concentrations ([Ca2+]i) even though Ca2+‐signalling pathways were intact as shown with vasopressin and endothelin. Exposure to 10 μM CdCl2 for 8 h nevertheless caused an increase in [Ca2+]i to > 250 nM and increases in [Ca2+]i achieved with ionophores alone were sufficient to decrease F‐actin concentrations. However, a rise in [Ca2+]i is not necessary for actin depolymerization. Depletion of cellular Ca2+ by treatment with thapsigargin did not protect F‐actin against Cd2+; the effect of Cd2+ was enhanced in cells unable to increase their [Ca2+]i. We conclude that depolymerization of F‐actin by Cd2+ in smooth muscle and mesangial cells is metal‐specific, Ca2+‐independent, and accompanied by a depletion of total actin protein. © 1996 Wiley‐Liss, Inc.

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The effects of pH and temperature on fluorescent calcium indicators as determined with Chelex-100 and EDTA buffer systems

Cited by 142 publications

References 12 publications

Role of cytosolic free calcium in the reorientation of pollen tube growth

Role of cytosolic free calcium in the reorientation of pollen tube growth

Increased perinuclear Ca2+ activity evoked by metabotropic glutamate receptor activation in rat hippocampal neurones.

Calcium-independent effects of cadmium on actin assembly in mesangial and vascular smooth muscle cells

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