FURA-2AM

Effects of transmitters and amyloid-beta peptide on calcium signals in rat cortical astrocytes: Fura-2AM measurements and stochastic model simulations.[Pubmed:21483471]

PLoS One. 2011 Mar 29;6(3):e17914.

BACKGROUND: To better understand the complex molecular level interactions seen in the pathogenesis of Alzheimer's disease, the results of the wet-lab and clinical studies can be complemented by mathematical models. Astrocytes are known to become reactive in Alzheimer's disease and their ionic equilibrium can be disturbed by interaction of the released and accumulated transmitters, such as serotonin, and peptides, including amyloid- peptides (A). We have here studied the effects of small amounts of A25-35 fragments on the transmitter-induced calcium signals in astrocytes by FURA-2AM fluorescence measurements and running simulations of the detected calcium signals. METHODOLOGY/PRINCIPAL FINDINGS: Intracellular calcium signals were measured in cultured rat cortical astrocytes following additions of serotonin and glutamate, or either of these transmitters together with A25-35. A25-35 increased the number of astrocytes responding to glutamate and exceedingly increased the magnitude of the serotonin-induced calcium signals. In addition to A25-35-induced effects, the contribution of intracellular calcium stores to calcium signaling was tested. When using higher stimulus frequency, the subsequent calcium peaks after the initial peak were of lower amplitude. This may indicate inadequate filling of the intracellular calcium stores between the stimuli. In order to reproduce the experimental findings, a stochastic computational model was introduced. The model takes into account the major mechanisms known to be involved in calcium signaling in astrocytes. Model simulations confirm the principal experimental findings and show the variability typical for experimental measurements. CONCLUSIONS/SIGNIFICANCE: Nanomolar A25-35 alone does not cause persistent change in the basal level of calcium in astrocytes. However, even small amounts of A25-35, together with transmitters, can have substantial synergistic effects on intracellular calcium signals. Computational modeling further helps in understanding the mechanisms associated with intracellular calcium oscillations. Modeling the mechanisms is important, as astrocytes have an essential role in regulating the neuronal microenvironment of the central nervous system.

Autofluorescence as a confound in the determination of calcium levels in hippocampal slices using fura-2AM dye.[Pubmed:8822368]

Brain Res. 1996 Jan 15;706(2):283-8.

Recent publications have reported calcium level determinations in slices of brain using imaging techniques and the dye FURA-2AM. In general these studies ignore or deal only perfunctorily with the problem of autofluorescence in slices. This confound, which is a result of the pyridine nucleotides that are normally present in tissue, has been previously reported to interfere with Ca2+ measurements in slices. Because these pyridine compounds are involved in cell metabolism, the fluorescence intensity is labile over time following experimental manipulations. We were studying Ca2+ levels in hippocampal slices using standard imaging techniques. We found significant and variable autofluorescence at the wavelengths used for calcium determination which interfered with data interpretation in fura-treated slices. The intensity of this autofluorescence is an additive effect and is not large enough to be observed when imaging monolayers. In this paper we present a method for conducting experiments and analyzing data that decreases interference from autofluorescence. Experiments were carried out on both slices bulk loaded with FURA-2AM and slices loaded with control buffer. A point to point subtraction of the control slice values gave representative calcium fluorescence values. Hippocampal slices were challenged with sodium cyanide or kainic acid. The metabolic response, seen in the fura-free slices, and the calcium response varied within and between these two treatments. Regional differences in the hippocampal sub fields were also demonstrated in response to the two treatments. These corresponded to known regional vulnerabilities to cyanide and kainate. We conclude that autofluorescence in slices need be considered when determining calcium concentrations using FURA-2AM.

Sildenafil inhibits human pulmonary artery smooth muscle cell proliferation by decreasing capacitative Ca2+ entry.[Pubmed:18818482]

J Pharmacol Sci. 2008 Sep;108(1):71-8.

Ca(2+) is a pivotal signal in human pulmonary artery smooth muscle cells (PASMCs) proliferation. Capacitative Ca(2+) entry (CCE) via the store-operated channel (SOC), which encoded by the transient receptor potential (TRP) gene, is an important mechanism for regulating intracellular Ca(2+) concentration ([Ca(2+)](i)) in PASMCs. Sildenafil, a potent type 5 nucleotide-dependent phosphodiesterase (PDE) inhibitor, has been proposed as a therapeutic tool to treat or prevent pulmonary arterial hypertension (PAH); however, the mechanism of its antiproliferative effect on PASMCs remains unclear. This study was designed to investigate the possible antiproliferative mechanism of sildenafil on human PASMCs, namely, its effect on the Ca(2+)-signal pathway. Cultured normal PASMCs were treated with endothelin-1 (ET-1) or ET-1 plus sildenafil separately. Cell number and viability were determined with a hemocytometer or MTT assay. [Ca(2+)](i) was measured by loading PASMCs with fura 2-AM. Expression of the TRPC1 gene and protein was detected by RT-PCR and Western blot, respectively. The results show that sildenafil dose-dependently inhibited the proliferation of PASMCs, the enhancement of basal [Ca(2+)](i) level, increase of CCE, and upregulation of TRPC expression induced by ET-1. These results suggest that sildenafil potently inhibits ET-1-induced PASMCs proliferation and downregulation of CCE and TRPC expression may be responsible for its antiproliferative effect.

A new generation of Ca2+ indicators with greatly improved fluorescence properties.[Pubmed:3838314]

J Biol Chem. 1985 Mar 25;260(6):3440-50.

A new family of highly fluorescent indicators has been synthesized for biochemical studies of the physiological role of cytosolic free Ca2+. The compounds combine an 8-coordinate tetracarboxylate chelating site with stilbene chromophores. Incorporation of the ethylenic linkage of the stilbene into a heterocyclic ring enhances the quantum efficiency and photochemical stability of the fluorophore. Compared to their widely used predecessor, "quin2", the new dyes offer up to 30-fold brighter fluorescence, major changes in wavelength not just intensity upon Ca2+ binding, slightly lower affinities for Ca2+, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca2+ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca2+, should make these dyes the preferred fluorescent indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues.