Thrombin-induced cytosolic alkalinization in human platelets occurs without an apparent involvement of HCO3-/Cl- exchange.
We have estimated the changes in cytosolic pH (pHi) that occur when human platelets are stimulated by thrombin. Changes in pHi were estimated (i) from the H+ efflux across the plasma membrane using an extracellular pH electrode and (ii) using an intracellular pH-sensitive fluorescent dye (BCECF). Stimulation of platelets with thrombin (0.5 unit/ml) resulted in an H+ efflux that averaged 7.7 +/- 1.6 mumol/10(11) platelets (means +/- SD) leading to an increase in pHi, from 7.05 +/- 0.04 to 7.45 +/- 0.05. Both H+ efflux and pHi changes were unaffected by 0.1 mM 4,4-diisothiocyanostilbene-2,2 disulphonate (DIDS), 0.1 mM 4'-acetamido 4'-isothiostilbene-2,2'-disulphonic acid (SITS), or 0.5 mM bumetanide, suggesting no involvement of anion transport systems, e.g. an HCO3-/Cl- exchange. Removal of HCO3- or Cl- from the suspending buffer had no effect on the extent of the rise in pHi. After blockade of Na+/H+ exchange by 100 microM ethylisopropylamiloride (EIPA), thrombin induced a decrease in pHi the rate of which averaged 0.39 unit/min in HCO3(-)-containing medium, and 0.57 unit/min in HCO3(-)-free medium. The cytosolic buffer capacity for H+ was determined by the nigericin/NH4Cl technique in BCECF-loaded platelets and averaged 25.3 mmol/(1xpH) in buffer containing 8 mM HCO3-, but only 17.2 mmol/(1xpH) in HCO3(-)-free buffer. The total amount of H+ transferred by Na+/H+ exchange can be estimated from our measurements at 10 mmol/l platelet cytosol in the absence of HCO3- and to 14 mmol/l platelet cytosol in the presence of HCO3-, and is in good agreement with the estimated amount of Na+ uptake by ADP-stimulated platelets.(ABSTRACT TRUNCATED AT 250 WORDS)
Clemens
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Institut für Physiologie, Ruhr-Universität, Bochum, Federal Republic of Germany.
Siffert
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Scheid
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D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D001639:Bicarbonates / Q000378:metabolism*; D001792:Blood Platelets / Q000378:metabolism*; D002034:Bumetanide / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013917:Thrombin / Q000494:pharmacology*
D001639:Bicarbonates; D002034:Bumetanide; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; C039465:5-dimethylamiloride; D000584:Amiloride; D013917:Thrombin; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid