Thrombin-induced cytosolic alkalinization in human platelets occurs without an apparent involvement of HCO 3 ? /Cl? exchange

Publisher Website
Google Scholar
Abstract
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 μmol/1011 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 HCO 3 − /Cl− exchange. Removal of HCO 3 − 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 μM ethylisopropylamiloride (EIPA), thrombin induced a decrease in pHi the rate of which averaged 0.39 unit/min in HCO 3 − -containing medium, and 0.57 unit/min in HCO 3 − -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 HCO 3 − , but only 17.2 mmol/(1xpH) in HCO 3 − -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 HCO 3 − and to 14 mmol/l platelet cytosol in the presence of HCO 3 − , and is in good agreement with the estimated amount of Na+ uptake by ADP-stimulated platelets. We conclude that net extrusion of H+ from stimulated platelets is predominantly mediated by Na+/H+ exchange without an apparent contribution of HCO 3 − /Cl− exchange.