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+]i. 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 muM 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 muM), whereas the effect of ouabain (20 and 50 muM), 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 muM 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.