Determination of Intracellular Ca2+ Concentration in the Human Pathogens Trypanosomatids Leishmania mexicana and Trypanosoma cruzi by the Use of the Fluorescent Ca2+ Indicator Fura-2

A large number of fluorescent calcium indicators are available for the determination of intracellular free calcium concentrations. The best known of these is fura-2. However, the employment of fura-2, amongst others, for the determination of intracellular free calcium is not problem-free.

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Axotomy induces a transient and localized elevation of the free intracellular calcium concentration to the millimolar range

Journal of Neurophysiology ◽

10.1152/jn.1995.74.6.2625 ◽

1995 ◽

Vol 74 (6) ◽

pp. 2625-2637 ◽

Cited By ~ 98

Author(s):

N. E. Ziv ◽

M. E. Spira

Keyword(s):

Intracellular Calcium ◽

Calcium Concentration ◽

Intracellular Calcium Concentration ◽

Concentration Gradients ◽

Fura 2 ◽

Direct Demonstration ◽

Free Intracellular Calcium ◽

Axonal Transection ◽

Transient Elevation

1. Axonal transection triggers a cascade of pathological processes that frequently lead to the degeneration of the injured neuron. It is generally believed that the degenerative process is triggered by an overwhelming influx of calcium through the cut end of the axon. 2. Theoretical considerations and indirect observations suggest that axotomy is followed by an increase in the free intracellular calcium concentration ([Ca2+]i) to the millimolar level. In contrast, only relatively modest and transient elevation in [Ca2+]i to the micromolar level was revealed by recent fura-2 studies. 3. In the current study we used the low-affinity Ca2+ indicator mag-fura-2 to reexamine the spatiotemporal distribution pattern of Ca2+ after axotomy and to map the free intracellular Mg2+ concentration gradients. 4. We report that axotomy elevates [Ca2+]i well beyond the "physiological" range of calcium concentrations, to levels > 1 mM near the tip of the cut axon and to hundreds of micromolars along the axon further away from the cut end. Nevertheless, [Ca2+]i recovers to the control levels within 2-3 min after the resealing of the cut end. 5. A comparison of the behavior of fura-2 and mag-fura-2 in the cytosol of the axotomized neurons reveals that the determination of [Ca2+]i by fura-2 largely underestimates the actual intracellular Ca2+ concentrations. 6. Experiments in which one branch of a bifurcated axon was transected revealed that the elevation in [Ca2+]i is confined to the transected axonal branch and does not spread beyond the bifurcation point. 7. After axotomy, the intracellular Mg2+ concentration equilibrates rapidly with the external concentration and then recovers at a rate somewhat slower than that of [Ca2+]i. 8. To the best of our knowledge, this study is the first direct demonstration that axotomy elevates [Ca2+]i to the millimolar range and that neurons are able to recover from these extreme calcium concentrations.

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Fura 2 determination of [Ca2+]i in isolated perfused heart using R wave-gated electromechanical shutters

Journal of Applied Physiology ◽

10.1152/jappl.1994.76.3.1394 ◽

1994 ◽

Vol 76 (3) ◽

pp. 1394-1399 ◽

Cited By ~ 8

Author(s):

D. P. Chen ◽

E. Jimenez ◽

K. Ataka ◽

S. Levitsky ◽

H. Feinberg

Keyword(s):

Cardiac Cycle ◽

Low Cost ◽

Rabbit Heart ◽

Isolated Perfused Heart ◽

Signal Averaging ◽

Perfused Heart ◽

Optical Components ◽

Fura 2 ◽

Heart Preparation

We describe a novel and relatively inexpensive spectrofluorescence system that supplies rapidly alternating wavelengths to either a standard cuvette or an isolated perfused heart. Its use is illustrated by determining changes in cytosolic intracellular Ca2+ concentration ([Ca2+]i) by using the Ca(2+)-sensitive fluorescent dye fura 2 in a rabbit heart preparation. The system uses two precision electromechanical shutters (capable of gating with respect to the electrocardiographic R wave for signal averaging) allowing alternate fura 2 excitation wavelengths (340 and 380 nm) without moving optical components and uses a fiber optic for conducting excitation and collecting epifluorescence. Sample recordings tracing the [Ca2+]i transient in an entire cardiac cycle and in capturing specific isolated regions (diastole and systole) of the cycle are presented. Limitations of this low-cost but easily implemented system are discussed.

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O-geranylchalcones: synthesis and metabolic inhibition against Leishmania mexicana and Trypanosoma cruzi

Medicinal Chemistry Research ◽

10.1007/s00044-019-02469-4 ◽

2019 ◽

Vol 29 (1) ◽

pp. 156-165

Author(s):

Karla Fabiola Chacon-Vargas ◽

Velvett G. Domínguez-Méndez ◽

Benjamín Nogueda-Torres ◽

David Chávez-Flores ◽

Alejandro A. Camacho-Dávila ◽

...

Keyword(s):

Trypanosoma Cruzi ◽

Metabolic Inhibition ◽

Leishmania Mexicana

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Determination of chemical structure and anti-Trypanosoma cruzi activity of extracts from the roots of Lonchocarpus cultratus (Vell.) A.M.G. Azevedo & H.C. Lima

Saudi Journal of Biological Sciences ◽

10.1016/j.sjbs.2020.08.036 ◽

2021 ◽

Vol 28 (1) ◽

pp. 99-108

Author(s):

Aline Antunes Maciel Bortoluzzi ◽

Izabela Virginia Staffen ◽

Fernanda Weyand Banhuk ◽

Aline Griebler ◽

Patricia Karoline Matos ◽

...

Keyword(s):

Trypanosoma Cruzi ◽

Chemical Structure

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Determination of intracellular Na and Ca in isolated guinea-pig atria by 23Na-NMK and fura-2 probe

The Japanese Journal of Pharmacology ◽

10.1016/s0021-5198(19)57501-1 ◽

1988 ◽

Vol 46 ◽

pp. 220

Author(s):

Yoshihiro Hotta ◽

Motoharu Nishiyama ◽

Hiroaki Ando ◽

Michio Yajima ◽

Kazumi Takeya ◽

...

Keyword(s):

Guinea Pig ◽

Fura 2 ◽

Guinea Pig Atria

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Free cytosolic Ca2+ measured with Ca(2+)-selective electrodes and fura 2 in rat mesenteric resistance arteries

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1993.265.2.h741 ◽

1993 ◽

Vol 265 (2) ◽

pp. H741-H746 ◽

Cited By ~ 4

Author(s):

P. E. Jensen ◽

M. J. Mulvany ◽

C. Aalkjaer ◽

H. Nilsson ◽

H. Yamaguchi

Keyword(s):

Dissociation Constant ◽

Isometric Force ◽

Force Measurements ◽

Resistance Arteries ◽

Fura 2 ◽

Good Accordance ◽

Dissociation Constant Kd

Free cytosolic Ca2+ was measured with sub-micrometer-tip, double-barrelled, Ca(2+)-selective electrodes and fura 2 in rat mesenteric resistance arteries. The purpose was to establish intracellular free Ca2+ concentration ([Ca2+]i) values in resting and stimulated vessels. Isolated vessels were mounted for isometric force measurements. Measured with electrodes, mean [Ca2+]i was 115 and 708 nM under resting and norepinephrine-activated conditions, respectively. Fura 2 was calibrated intracellularly including determination of the intracellular dissociation constant (Kd) of the fura 2:Ca2+ complex. The intracellular Kd was 342 nM. With this value of Kd, fura 2 measurements of mean [Ca2+]i were 129 and 537 nM under resting and norepinephrine-activated conditions, respectively. The values measured with the two techniques were thus in good accordance.

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