Thrombin-induced cytosolic alkalinization in human platelets occurs without an apparent involvement of HCO3−/Cl−exchange
✍ Scribed by Norbert Clemens; Winfried Siffert; Peter Scheid
- Book ID
- 104744757
- Publisher
- Springer
- Year
- 1990
- Tongue
- English
- Weight
- 667 KB
- Volume
- 416
- Category
- Article
- ISSN
- 0031-6768
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✦ Synopsis
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 + effiux 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/~mol/10 I1 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 4,4diisothiocyanostilbene-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/C1-exchange. Removal of HCOy or C1-from the suspending buffer had no effect on the extent of the rise in pHi. After blockade of Na+/H + exchange by 100 ~tM ethylisopropylamiloride (EIPA), thrombin induced a decrease in pHi the rate of which averaged 0.39 unit/rain in HCO3-containing medium, and 0.57 unit/min in HCO~-free medium. The cytosolic buffer capacity for H + was determined by the nigericin/ NH4C1 technique in BCECF-loaded platelets and averaged 25.3 mmol/(lxpH) in buffer containing 8 mM HCO~, but only 17.2 mmol/(lxpH) in HCO;--free buffer. The total amount of H + transferred by Na+/H + exchange can be estimated from our measurements at 10 mmol/1 platelet cytosol in the absence of HCO3 and to 14 retool/1 platelet cytosol in the presence of HCO3, 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 HCOJC1 exchange.