Detection of intracellular free Na+ concentration of synaptosomes by a fluorescent indicator, Na(+)-binding benzofuran isophthalate: the effect of veratridine, ouabain, and alpha-latrotoxin.
A novel fluorescent Na+ indicator, Na(+)-binding benzofuran isophthalate (SBFI), was used to follow changes in the intracellular free Na+ concentration ([Na+]i) of synaptosomes. The dye, when loaded into synaptosomes in the form of its acetoxymethyl ester, was responsive to changes of [Na+]. Calibration was made using the 340/380 nm excitation ratio when the cytoplasmic Na+ concentration was equilibrated with different concentrations of extracellular Na+ in the presence of 2 microM gramicidin D. The basal value of [Na+]i in synaptosomes in the presence of 140 mM extracellular Na+ was found to be 10.9 +/- 1.8 mM. Veratridine, which opens potential-dependent Na+ channels, caused a sudden increase in [Na+]i in a concentration-dependent manner (1-20 microM), whereas the effect of ouabain (20 and 50 microM), the inhibitor of the plasma membrane Na+,K(+)-ATPase, was more gradual. The rise in the fluorescence intensity upon addition of veratridine was prevented completely by 2 microM tetrodotoxin. alpha-Latrotoxin, the black widow spider toxin, caused an increase in the fluorescence intensity, which became evident 1 min after the addition of the toxin. The rate of increase was proportional to the concentration of the toxin (0.19-1.5 nM). This report confirms our earlier finding demonstrating a Na(+)-dependent component in the action of alpha-latrotoxin, and shows that changes in [Na+]i in synaptosomes can be followed by SBFI.
Deri
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Department of Biochemistry II, Semmelweis University of Medicine, Budapest, Hungary.
Adam-Vizi
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D000818:Animals; D001572:Benzofurans; D002138:Calibration; D004988:Ethers, Cyclic; D005456:Fluorescent Dyes; D006168:Guinea Pigs; D007425:Intracellular Membranes / Q000378:metabolism*; D009994:Osmolar Concentration; D010042:Ouabain / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism*; D013111:Spider Venoms / Q000494:pharmacology; D013574:Synaptosomes / Q000378:metabolism*; D014701:Veratridine / Q000494:pharmacology
D001572:Benzofurans; D004988:Ethers, Cyclic; D005456:Fluorescent Dyes; D013111:Spider Venoms; C061665:sodium-binding benzofuran isophthalate; D010042:Ouabain; C020763:alpha-latrotoxin; D014701:Veratridine; D012964:Sodium