Stimulation by thrombin increases the cytosolic free Na+ concentration in human platelets. Studies with the novel fluorescent cytosolic Na+ indicator sodium-binding benzofuran isophthalate.
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.
Borin
M
M
Max-Planck-Institut für Biophysik, Frankfurt/Main, Federal Republic of Germany.
Siffert
W
W
D000527:Alprostadil / Q000494:pharmacology; D001572:Benzofurans; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002352:Carrier Proteins / Q000097:blood; D000242:Cyclic AMP / Q000097:blood; D003600:Cytosol / Q000378:metabolism; D004988:Ethers, Cyclic; D005456:Fluorescent Dyes; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D008985:Monensin / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology; D015539:Platelet Activation / Q000502:physiology; D011422:Propionates / Q000494:pharmacology; D012964:Sodium / Q000097:blood*; D017923:Sodium-Hydrogen Exchangers; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000097:blood; D013050:Spectrometry, Fluorescence; D013917:Thrombin / Q000494:pharmacology*
D001572:Benzofurans; D002352:Carrier Proteins; D004988:Ethers, Cyclic; D005456:Fluorescent Dyes; D011422:Propionates; D017923:Sodium-Hydrogen Exchangers; C061665:sodium-binding benzofuran isophthalate; D015759:Ionomycin; D010042:Ouabain; D008985:Monensin; D012964:Sodium; D000242:Cyclic AMP; D013917:Thrombin; D000254:Sodium-Potassium-Exchanging ATPase; D000527:Alprostadil; C029658:propionic acid