Identification of a sodium-bicarbonate symport in human platelets

Abstract

Intracellular pH (pH i) was measured in human platelets using fluorescent probes. Basal pH i was higher in HCO 3 −-buffered solutions (7.33 ± 0.01) than in nominally HCO 3 − 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 pH i when platelets maintained in Hepes were transferred to a CO 2 HCO 3 − buffer. After an intracellular acidosis induced by an NH 4Cl prepulse, the initial velocity of recovery ( d(pH) dt i , in pH units/min) was 3.32 ± 0.69 in Hepes-buffered solution and 2.85 ± 0.88 in HCO 3 − 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 μ mol 1 EIPA effectively blocked acid efflux (d(pH)/d t i = 0.08 ± 0.04) in a nominally HCO 3 −-free solution, whereas the recovery was reduced but not abolished (d(pH)/d t i = 0.37 ± 0.10, P < 0.05) in the presence of bicarbonate. The stilbene derivative SITS further inhibited the EIPA-resistant pH i recovery. Removal of external Na + inhibited the HCO 3 −-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 + HCO 3 − cotransport in the recovery of pH i following an acid load. Both the Na + H + antiport and the HCO 3 −-dependent mechanism contribute approx. 50% each to the total acid equivalent efflux during the recovery from a pH i6.46 ± 0.14 to the basal pH i in human platelets.