Stimulation by thrombin increases the cytosolic free Na+ concentration in human platelets. Studies with the novel fluorescent cytosolic Na+ indicator sodium-binding benzofuran isophthalate

J Biol Chem. 1990 Nov 15;265(32):19543-50.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alprostadil / pharmacology
  • Benzofurans*
  • Blood Platelets / drug effects
  • Blood Platelets / metabolism*
  • Carrier Proteins / blood
  • Cyclic AMP / blood
  • Cytosol / metabolism
  • Ethers, Cyclic*
  • Fluorescent Dyes
  • Humans
  • Ionomycin / pharmacology
  • Monensin / pharmacology
  • Ouabain / pharmacology
  • Platelet Activation / physiology
  • Propionates / pharmacology
  • Sodium / blood*
  • Sodium-Hydrogen Exchangers
  • Sodium-Potassium-Exchanging ATPase / antagonists & inhibitors
  • Sodium-Potassium-Exchanging ATPase / blood
  • Spectrometry, Fluorescence
  • Thrombin / pharmacology*


  • Benzofurans
  • Carrier Proteins
  • Ethers, Cyclic
  • Fluorescent Dyes
  • Propionates
  • Sodium-Hydrogen Exchangers
  • sodium-binding benzofuran isophthalate
  • Ionomycin
  • Ouabain
  • Monensin
  • Sodium
  • Cyclic AMP
  • Thrombin
  • Sodium-Potassium-Exchanging ATPase
  • Alprostadil
  • propionic acid