Identification of a sodium-bicarbonate symport in human platelets

AbstractIntracellular pH (pHi) was measured in human platelets using fluorescent probes. Basal pHi was higher in HCO3−-buffered solutions (7.33 ± 0.01) than in nominally HCO3− free, Hepes-buffered solutions (7.16 ± 0.01, P < 0.05). Addition of EIPA caused a fall in Hepes, but did not inhibit the increase of pHi when platelets maintained in Hepes were transferred to a CO2HCO3− buffer. After an intracellular acidosis induced by an NH4Cl prepulse, the initial velocity of recovery (d(pH)dti, in pH units/min) was 3.32 ± 0.69 in Hepes-buffered solution and 2.85 ± 0.88 in HCO3− media. Taking into account the differences in buffer capacity, the efflux of acid equivalents after 1.2 min was twice as much in the presence of bicarbonate. The addition of 30 μmol1 EIPA effectively blocked acid efflux (d(pH)/dti = 0.08 ± 0.04) in a nominally HCO3−-free solution, whereas the recovery was reduced but not abolished (d(pH)/dti = 0.37 ± 0.10, P < 0.05) in the presence of bicarbonate. The stilbene derivative SITS further inhibited the EIPA-resistant pHi recovery. Removal of external Na+ inhibited the HCO3−-dependent recovery whereas depletion of internal Cl−, did not suppress it. Depolarization of the membrane had no effect on this recovery. The results suggest the contribution of an electroneutral Na+HCO3− cotransport in the recovery of pHi following an acid load. Both the Na+H+ antiport and the HCO3−-dependent mechanism contribute approx. 50% each to the total acid equivalent efflux during the recovery from a pHi6.46 ± 0.14 to the basal pHi in human platelets