Anatoxin-a (ATX) is a potent neurotoxin produced by several species of Anabaena spp. Cyanobacteria blooms around the world have been increasing in recent years; therefore, it is urgent to develop sensitive techniques that unequivocally confirm the presence of these toxins in fresh water and cyanobacterial samples. In addition, the identification of different ATX analogues is essential to later determine its toxicity. In this paper we designed a fluorescent polarization (FP) method to detect ATXs in water samples. A nicotinic acetylcholine receptor (nAChR) labeled with a fluorescein derivative was used to develop this assay. Data showed a direct relationship between the amount of toxin in a sample and the changes in the polarization degree of the emitted light by the labeled nAChR, indicating an interaction between the two molecules. This method was used to measure the amount of ATX in three Anabaena spp. cultures. Results indicate that it is a good method to show ATXs presence in algal samples. In order to check the toxin profile of Anabaena cultures a LC-MS/MS method was also developed. Within this new method, ATX-a, retention time (RT) 5 min, and three other molecules with a mass m/z 180.1 eluting at 4.14 min, 5.90 min and 7.14 min with MS/MS spectra characteristic of ATX toxin group not previously identified were detected in the Anabaena spp. cultures. These ATX analogues may have an important role in the toxicity of the sample.

Neurotoxin

Marine toxin

Cylindrospermopsin

10.3390/toxins6020402

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Characterization of the Na<sup>+</sup>/Ca<sup>2+</sup> Exchanger on Rat Mast Cells

Cellular Physiology and Biochemistry (1999)

Amparo Alfonso Jorge Lago M.A. Botana Mercedes R. Vieytes Luís M. Botana

The Na+/Ca2+ exchanger has not been characterized in rat mast cells, although its exitstence has been previously suggested. In this work, we determine that this exchanger exists on rat mast cells and that it has an important regulatory role on the cellular function. We have studied uptake and release of 45Ca in the presence of different external sodium concentrations and, under the same conditions, the simultaneous uptake of 22Na and 45Ca. The results show that uptake and release of 45Ca in these cells are related to the concentration of sodium in the extracellular medium and that there is also a perfect coupling between 22Na and 45Ca fluxes. In these conditions, we evaluated the intracellular calcium levels in fura-2 loaded cells. When the extracellular sodium concentration was lower than 60 mM, we observed an increase in intracellular calcium, reaching its maximum when the extracellular medium has no sodium. Then we investigated the effect on histamine release. The ionophore A23187 elicits histamine release in rat mast cells, depending on the extracellular calcium concentration. This drug releases more histamine (up to twofold) with sodium concentrations <60 mM. In the presence of 2,4-dichlorobenzamil hydrochloride, a Na+/Ca2+ exchanger inhibitor, the release of histamine induced by the ionophore was lower than in controls in media with low external sodium, and, on the contrary, at extracellular sodium concentrations >60 mM, the histamine release was higher than in controls. In the same conditions, but when the Na+-K+ ATPase was inhibited by ouabain, and as a consequence more sodium was inside the cells, a high increase in histamine release induced by A23187 in a sodium-free medium was observed. Under the same conditions, a high increase in intracellular calcium takes place. The overall data are preliminary evidence suggesting the existence of a Na+/Ca2+ exchanger in rat mast cells with a threshold to get reversed at 60 mM external sodium, lower concentrations of this ion increasing internal calcium and producing higher histamine release.

Sodium-calcium exchanger

10.1159/000016302

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Citations (11)