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Abstract

The new fluorescent Na⁺ indicator sodium-binding benzofuran isophthalate (SBFI) was used for determination of the cytosolic free Na⁺ concentration, [Na⁺]i, in human platelets. The dye could be loaded into platelets in the form of its acetoxymethyl ester (SBFI-AM). Calibration of the fluorescence in terms of [Na⁺]_i was done by measuring the 345/385 nm excitation ratio (emission 490 nm) at various extracellular Na⁺ concentrations, [Na⁺]_o, in the presence of gramicidin D. The 345/385 intensity ratio increased almost linearly when [Na⁺]_i was stepwise raised from 20 to 60 mM. The basal value for [Na⁺]_i was found to be 26.0 +/- 4.5 mM (n = 15). Incubation of platelets in Na⁺-free buffer decreased [Na⁺]_i, whereas inhibition of the (Na⁺ + K⁺)-ATPase by 0.5 mM ouabain increased [Na⁺]_i to 56 +/- 4 mM (n = 4) within 60 min. Activation of Na⁺/H⁺ exchange by exposing platelets to propionic acid also raised [Na⁺]_i, and a comparable effect was produced by the Na⁺/H⁺ ionophore monensin. Activation of platelets with thrombin (0.1-0.5 unit/ml) also increased the 345/385 nm intensity ratio, an effect that was not seen in Na⁺-free buffer or after raising intracellular cAMP by treatment of platelets with prostaglandin E₁. On the average, [Na⁺]_i was raised to 59.5 +/- 5.3 mM (n = 15) at 10 min after addition of thrombin without a significant decrease for further 10 min. An increase in [Na⁺]_i was also seen when platelets were challenged with the Ca²⁺ ionophore ionomycin, an effect that did not occur in the absence of Na⁺_o. Our findings confirm earlier reports which demonstrated a rise in [Na⁺]_i in stimulated platelets and show that SBFI is a useful tool for determination of [Na⁺]_i in resting and stimulated platelets.